Method of treating soy proteins and soy protein product produced by this method

ABSTRACT

A method of treating a proteinaceous material having a first concentration of β-conglycinin, the method including combining the proteinaceous material with an enzyme to form a reaction mixture, the reaction mixture initially having a pH of at least about 7.0 standard pH units, allowing the enzyme to hydrolyze β-conglycinin present in the reaction mixture to form a proteinaceous intermediate, and inactivating the enzyme present in the reaction mixture after a reaction period to form a proteinaceous product, the proteinaceous product having a second concentration of β-conglycinin, the second concentration of β-conglycinin being at least 99 percent less than the first concentration of β-conglycinin.

CROSS-REFERENCE TO RELATED APPLICATION(S)

[0001] This application claims priority from U.S. Patent ApplicationSerial No. 60/199,758 that was filed on Apr. 26, 2000.

BACKGROUND OF THE INVENTION

[0002] The present invention generally relates to a method of reducingthe antigenicity of vegetable proteins, while also improving thesolubility characteristics of the vegetable proteins. More particularly,the present invention relates to a method of enzymatically hydrolyzingvegetable proteins, such as raw, natural soy proteins and denatured soyproteins, to reduce the antigenicity of the vegetable proteins whilealso improving the solubility characteristics of the vegetable proteins.

[0003] Over the years, researchers have found that soybeans may beprocessed to recover or extract a number of valuable components, such assoy protein and soybean oil, from the soy beans. Also, soybeans may beprocessed to form soy flours high in nutritionally beneficialsubstances, such as fiber and protein. Such processing of soybeans ofteninclude heat treatment for a variety of purposes, such as inactivatingdestructive enzymes or inactivating compounds responsible foroff-flavors that are unpalatable to humans and/or animals.

[0004] Soybean processing techniques that employ heat frequently causedenaturation of proteins present in resulting soy component andproducts. The degree of protein denaturation depends upon the durationof heat and the temperature profile during the heating, among otherfactors. Additionally, some proteins in soybeans are more susceptible todenaturation at particular heating conditions than are other soybeanproteins. Nonetheless, denaturation of soy proteins is problematic sincedenatured proteins typically exhibit greatly diminished solubility inwater and aqueous solutions.

[0005] Many soy products, such as soy flour, soy flakes, and soy meal,are available and are commonly used for production of animal feeds andfood products for human consumption. However, any such soy products thathave been heat processed to a substantial degree have also undergonesubstantial soy protein denaturation and, consequently, frequently havea Protein Dispersability Index (subsequently referred to as “PDI”) onthe order of about 20 or even less. The PDI is a measure of proteinsolubility (and consequently a measure of protein dispersability) inwater. The PDI decreases as the level of protein denaturation in a soycomponent or product increases, absent further processing of thedenatured protein to enhance the solubility of the denatured protein.Though there are vegetable protein products with relative high PDIs of90 or more, and thus high levels of soluble proteins, these products aretypically very expensive and/or often contain high levels of antigenicproteins.

[0006] Heat treating of soybeans and soybean components, althoughbeneficial for deactivating destructive enzymes and compounds thatcontribute to unpalatable tastes, nevertheless do little, if anything,to reduce the antigenicity of the heat-processed soybean products. Theantigenicity of a particular substance is directly correlated to theconcentration of antigens present in the substance. Glycinin andβ-conglycinin, which are commonly referred to as antigenic proteins, aretwo proteins in soybean products that cause the majority of theantigenicity typically observed in soybean products. Consequently,glycinin and β-conglycinin, by their presence or absence, predominantlycontrol the level of antigenicity of a particular soybean product.

[0007] Heat-treating and heat-processing typically do not sufficientlyreduce the concentration of antigenic proteins, such as glycinin andβ-conglycinin, in a particular proteinaceous material. Other soybeanprocessing techniques exist that may or may not incorporate heattreatment steps. For example, some commercial processing plants employorganic solvents, such as hexane, to extract oil from soy beans or soyproducts, such as soy flakes. The heat that is applied during the oilextraction process causes some denaturation of protein in the soyproducts. The heat is typically employed during the oil extractionprocess for purposes of evaporating the organic solvent. This heatingfor solvent evaporation purposes may cause some reduction of theantigenic protein concentration, though any such reduction is only aninsignificant reduction. The organic solvent, such as hexane, that isemployed in these processes for oil extraction purposes typically doesnot cause the destruction or removal of antigenic proteins, such asglycinin and P-conglycinin. There are other organic solvents that may beemployed in these processes for purposes other than oil extraction. Someof these other organic solvents may even bring about significantreductions of the concentration of antigenic proteins, such as glycininand β-conglycinin, in a particular proteinaceous material.

[0008] The destruction of antigenic protein that provides a reducedlevel of antigenicity in soybean products is important, since antigens,such as antigenic proteins, when introduced into a human being or intoan animal, frequently cause production of antibodies that lead todevelopment of allergic reactions that in turn reduce the digestibilityof soybean products or cause other nutritional disturbances. Thus, toreduce the opportunity for allergic reactions, it is beneficial toreduce the antigenicity of soybean products by reducing theconcentration of antigenic proteins, such as glycinin and P-conglycinin,in the soybean products.

[0009] However, soybean processing techniques that rely on organicsolvents, even though beneficial for destruction of antigenic proteins,are not an optimum solution to the antigenicity issue. First, reducingthe antigenicity of soybean products using such solvent-based processingtechniques nevertheless typically leaves the soybean products with highlevels of denatured proteins. These high levels of denatured proteinscontribute to poor protein solubility characteristics in soybeanproducts produced by solvent-based processing techniques. Furthermore,complete removal of the organic solvent from soybean products producedby solvent-based processing techniques is challenging and oftenincomplete, since trace levels of the organic solvent typically remainin the soybean product. Consumers are increasingly aware of researchstudies that raise questions about the effects of trace levels oforganic solvents on human health. Therefore, to raise public perceptionof food quality, it is useful to minimize or even eliminate use oforganic solvents in food processing techniques.

[0010] However, other than solvent-based processing techniques,heat-based processing techniques that denature proteins while leavingantigenic proteins intact or substantially intact are the most commonsoybean processing techniques. Furthermore, other processing techniques,such as grinding or milling, though not relying upon heating thatdenatures proteins, nevertheless, typically leave high and substantiallevels of antigenic protein in the processed soybean components.

[0011] Thus, there is a need in the food and animal feed manufacturingindustries for a technique of processing vegetable protein sources, suchas soybeans and soybean components, that reduces the antigenicity insoybean products to reduce the potential for allergic reactions inhumans and animals that consume the soybean products. Furthermore, thereis a need for a food and animal feed processing technique that improvesthe solubility, and thus the dispersability, of denatured proteins invegetable sources of protein, such as soybean products. Enhancedsolubility and dispersability of denatured proteins is necessary toallow production of beverages, such as milk substitutes, milk replacers,and infant formulas, that contain proteins derived from vegetablesources, such as soybeans, and to support production of food productsand animal feeds that incorporate dispersed or emulsified proteinsderived from vegetable sources, such as soybeans. The process of thepresent invention provides an optimum solution to these needs byproviding a product with proteins exhibiting high levels of solubilitywhere the product also contains minimal, if any, levels of antigenicproteins.

BRIEF SUMMARY OF THE INVENTION

[0012] The present invention includes a method of treating aproteinaceous material having a first concentration of β-conglycinin.The method includes combining the proteinaceous material with an enzymeto form a reaction mixture, the reaction mixture initially having a pHof at least about 7.0 standard pH units, allowing the enzyme tohydrolyze β-conglycinin present in the reaction mixture to form aproteinaceous intermediate, and inactivating the enzyme present in thereaction mixture after a reaction period to form a proteinaceousproduct. The proteinaceous product produced by the method has a secondconcentration of β-conglycinin that is at least 99 percent less than thefirst concentration of β-conglycinin. The present invention alsoincludes a method of treating a proteinaceous material, a method oftreating a proteinaceous material having a first concentration ofglycinin, and a method of treating a proteinaceous material having afirst Protein Dispersability Index.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1 is a size distribution plot of protein fragments withdifferent molecular weights present in a vegetable protein source thatwas used as feed material in the process of the present invention.

[0014]FIG. 2 is a size distribution plot of protein fragments withdifferent molecular weights present in a vegetable protein productproduced by the process of the present invention based upon the feedmaterial of FIG. 1.

[0015]FIG. 3 is a plot of pH and viscosity profiles of a pair ofdifferent slurries based on vegetable protein sources during enzymatichydrolysis of the slurries in accordance with the present invention.

[0016]FIG. 4 is a size distribution plot of protein fragments withdifferent molecular weights present in another vegetable protein sourcethat was used as feed material in the process of the present invention.

[0017]FIG. 5 is a size distribution plot of protein fragments withdifferent molecular weights present in a vegetable protein productproduced by the process of the present invention based upon the feedmaterial of FIG. 4.

DETAILED DESCRIPTION

[0018] The present invention generally relates to a method of reducingthe antigenicity of vegetable proteins, while also improving thesolubility characteristics of the vegetable proteins. More particularly,the present invention relates to a method of enzymatically hydrolyzingvegetable proteins, such as raw, natural soy proteins and denatured soyproteins, to reduce the antigenicity of the vegetable proteins whilealso improving the solubility characteristics of the vegetable proteins.

[0019] Briefly, the process of the present invention entails theformation of an aqueous slurry of one or more vegetable proteinmaterials to form a slurried vegetable protein feed. The slurriedvegetable protein feed is subjected to the action of a protease (a“proteolytic enzyme”) to produce a slurried vegetable protein product.The pH of the slurried vegetable protein feed and the temperature of theslurried vegetable protein feed that are selected such that action ofthe protease on proteins present in the slurried vegetable protein feedis effective (1) to enhance the level of protein solubility in theslurried vegetable protein product, as compared to the level of proteinsolubility in the slurried vegetable protein feed, and (2) to reduce thelevel of antigenicity in the slurried vegetable protein product, ascompared to the level of antigenicity in the slurried vegetable proteinfeed. Preferably, the slurried vegetable protein feed, at an alkalinepH, is subjected to the action of an alkaline proteolytic enzyme at atemperature of about 60° C. or less, to produce the slurried vegetableprotein product. The slurried vegetable protein product, afterpreparation, is then heated to inactivate the proteolytic enzyme and isthereafter dried to form a powdered vegetable protein product of thepresent invention.

[0020] The process of the present invention may be beneficially employedto hydrolyze proteins from any source, such as vegetable proteinmaterials, animal protein materials, marine protein materials, and anycombination of any of these. Some examples of vegetable proteinmaterials are protein materials derived from soybeans, such as soyprotein isolate, toasted or untoasted soy flour, soy grits, soy flakes,soy meal, soy protein concentrates, and any combination of any of these.The vegetable protein material, such as any of the soybean proteinsources listed above, maybe defatted, reduced fat, or full fat vegetableprotein materials. Some examples of animal protein materials include eggalbumin isolate; powdered egg whites; dairy protein materials, such aswhey protein isolate, whey protein concentrate, and powdered whey; andany combination of any of these. As with the vegetable proteinmaterials, any animal protein material(s) may be defatted, reduced fat,or full fat in nature. Some examples of marine protein materials includeprotein-containing materials derived from marine creatures, such asfish. As with the vegetable protein materials and the animal proteinmaterials, any marine protein material(s) may be defatted, reduced fat,or full fat in nature.

[0021] Though descriptions of the present invention are primarily madein terms of vegetable protein material, it is to be understood that anyother protein material, such as animal protein materials and marineprotein materials, may be substituted in place of vegetable proteinmaterial, in accordance with the present invention, while stillrealizing benefits of the present invention. Likewise, it is to beunderstood that any combination of any protein material, such as anycombination of vegetable protein material, animal protein materials,and/or marine protein materials, may be processed in accordance with thepresent invention, while still realizing benefits of the presentinvention.

[0022] The slurried vegetable protein feed may be prepared by combiningthe vegetable protein material with water. While the total solidsconcentration in the slurried vegetable protein feed is not critical tothe present invention, the total solids concentration in the slurriedvegetable protein feed preferably ranges between about 10 weight percentand about 35 weight percent, based upon the total weight of the slurriedvegetable protein feed. Total solids concentrations higher than about 35weight percent are less desirable because such higher concentrationsincrease the viscosity of the slurried vegetable protein feed andconsequently may cause difficulties in preparing, mixing and/or handlingthe slurried vegetable protein feed. Total solids concentrations lowerthan about 10 weight percent in the slurried vegetable protein feed areless preferred, because such lower total solids concentrations wouldincrease the size of equipment needed to accomplish the process of thepresent invention and would ultimately require removal of greateramounts of moisture to produce the powdered vegetable protein product ofthe present invention.

[0023] After preparation, the temperature of the slurried vegetableprotein feed is adjusted to a temperature where action of the proteaseon proteins present in the slurried vegetable protein feed is effective(1) to enhance the level of protein solubility in the slurried vegetableprotein product, as compared to the level of protein solubility in theslurried vegetable protein feed, and (2) to reduce the level ofantigenicity in the slurried vegetable protein product, as compared tothe level of antigenicity in the slurried vegetable protein feed.Preferably, when the protease is an alkaline proteolytic enzyme, theslurried vegetable protein feed is heated to a temperature of about 60°C., or less, such as to a temperature of about 50° C. to about 60° C.

[0024] The slurried vegetable protein feed may be held in a batchreactor, such as a tank or other vessel that is jacketed for circulationof steam, hot water, or other heating fluid to attain and maintain thedesired temperature, such as the preferred temperature of about 60° C.,or less. Alternatively, the slurried vegetable protein feed may becirculated from the batch reactor, through a heat exchanger, and backinto the batch reactor to heat the slurried vegetable protein feed. Asanother alternative, the water that is blended with the vegetableprotein material to form the slurried vegetable protein feed maybeheated prior to combination of the vegetable protein material and thewater. The batch reactor containing the slurried vegetable protein feedshould be equipped with an agitator that is capable of maintaining thehomogeneity of the slurried vegetable protein feed during preparation,pH adjustment, and enzymatic hydrolysis.

[0025] After the slurried vegetable protein feed has been heated to thedesired temperature, such as the preferred temperature of about 60° C.,or less, an alkaline agent or an acidic agent, as appropriate, is addedto adjust the pH of the slurried vegetable protein feed. The pH of theslurried vegetable protein feed is adjusted to a pH that is within therange of pHs where action of the protease on proteins present in theslurried vegetable protein feed is effective (1) to enhance the level ofprotein solubility in the slurried vegetable protein product, ascompared to the level of protein solubility in the slurried vegetableprotein feed, and (2) to reduce the level of antigenicity in theslurried vegetable protein product, as compared to the level ofantigenicity in the slurried vegetable protein feed.

[0026] After the slurried vegetable protein feed has been heated to thedesired temperature, such as the preferred temperature of about 60° C.,or less, the alkaline agent is preferably added to adjust the pH of theslurried vegetable protein feed to a pH of about 7.0 standard pH units,or more, such as to a pH of about 7.0 standard pH units to about 10.0standard pH units, since the activity of one preferred alkalineproteolytic enzyme is improved within this pH range. More preferably,the pH of the slurried vegetable protein feed is adjusted to a pH ofabout 8.5 standard pH units, or more, such as to a pH of above about 8.5standard pH units to a pH of about 9.5 standard pH units, since theenzymatic hydrolysis reaction has been observed to enhance proteinsolubility and/or minimize antigenicity levels when the pH of theslurried vegetable protein feed is adjusted to this more preferredrange. Still more preferably, the pH of the slurried vegetable proteinfeed is adjusted to a pH ranging from about 9.0 standard pH units toabout 9.5 standard pH units, such as at a pH of about 9 standard pHunits, since the enzymatic hydrolysis reaction has been observed toenhance protein solubility and/or minimize antigenicity levels when thepH of the slurried vegetable protein feed is adjusted to this level.

[0027] The alkaline agent is preferably an edible, food grade alkalineagent. Some examples of suitable edible, food grade, alkaline agentsinclude sodium hydroxide, potassium hydroxide, calcium hydroxide, andmagnesium hydroxide. Typically, any alkaline agent that is used will bein the form of an aqueous solution of the alkaline agent, such as analkaline, aqueous solution containing about 10 weight percent of thealkaline agent in water, based upon the total weight of the aqueoussolution, to minimize the potential for over-shooting the desired pH ofthe slurried vegetable protein feed.

[0028] The acidic agent is preferably an edible, food grade acidicagent. Some examples of suitable edible, food grade, acidic agentsinclude hydrochloric acid and acetic acid. Typically, any acidic agentthat is used will be in the form of an aqueous solution of the acidicagent, such as an acidic, aqueous solution containing about 10 weightpercent of the acidic agent in water, based upon the total weight of theaqueous solution, to minimize the potential for over-shooting thedesired pH of the slurried vegetable protein feed.

[0029] After preparation and pH adjustment of the slurried vegetableprotein feed, the slurried vegetable protein feed is hydrolyzed tocleave proteins of the slurried vegetable protein feed into proteinfragments (peptides) with smaller molecular weights than the proteins ofthe slurried vegetable protein feed and to reduce the concentration ofantigenic proteins, such as glycinin and P-conglycinin, originallypresent in the slurried vegetable protein feed. The hydrolysis maybeachieved in a single stage enzymatic hydrolysis reaction that employsone or more proteolytic enzymes. Preferably, the one or more proteolyticenzymes are one or more alkaline proteolytic enzymes.

[0030] When the preferred alkaline proteolytic enzyme(s) is employed,the enzymatic hydrolysis reaction begins with the slurried vegetableprotein feed at a pH of about 7.0 standard pH units, such as at a pH ofabout 7.0 standard pH units to about 10.0 standard pH units; morepreferably with the slurried vegetable protein feed at a pH of about 8.5standard pH units, or more, such as at a pH above about 8.5 standard pHunits to about 9.5 standard pH units; more preferably with the slurriedvegetable protein feed at a pH within the range of from about 9.0standard pH units to about 9.5 standard pH units, such as at a pH ofabout 9 standard pH units. After the enzymatic hydrolysis reactionbegins, the pH of the slurried vegetable protein feed is thereafterpreferably allowed to freely change, without any subsequent pHadjustment or pH control. Changes in the pH of the slurried vegetableprotein feed are thought to be caused by the enzymatic hydrolysisreaction.

[0031] As an optional alternative, as the enzymatic hydrolysis reactionprogresses, the pH of the slurried vegetable protein feed may beadjusted or controlled to remain within the pH range or at the pH of theslurried vegetable protein feed that existed upon initiation of theenzymatic hydrolysis reaction. Preferably, however, such adjustment orcontrol of the pH is not done during the enzymatic hydrolysis reactionbecause (1) such pH adjustment or control may require additional laborand/or equipment, (2) such pH adjustment or control does notsignificantly affect (a) the beneficial protein solubility enhancementor (b) the beneficial antigenicity reduction that are achieved by theprocess of the present invention. Furthermore, such pH adjustment orcontrol does not significantly affect the rates at which the beneficialprotein solubility enhancement or the beneficial antigenicity reductionare achieved by the process of the present invention minimization.However, despite not needing to control pH during the enzymatichydrolysis reaction, the temperature of the slurried vegetable proteinfeed within the batch reactor is maintained at the desired reactiontemperature, such as the preferred reaction temperature of about 60° C.,or less, and agitation is maintained to maintain homogeneity of thecontents of the batch reactor during the enzymatic hydrolysis reaction.

[0032] Following addition of the proteolytic enzyme, such as thepreferred alkaline proteolytic enzyme, the enzymatic hydrolysis reactionis allowed to proceed at the selected temperature, such as the preferredtemperature of about 60° C., or less, for a period of time that iseffective to modify the proteinaceous components of the slurriedvegetable protein feed in accordance with the present invention and tothe desired degree. Though those of ordinary skill in the art willrecognize that this period of time may vary, depending upon theparticular proteolytic enzyme(s) employed, the activity of theproteolytic enzyme(s), the temperature of the slurried vegetable proteinfeed at the onset of, and during, the enzymatic hydrolysis reaction, andother factors, this period of time will, nevertheless, typically rangefrom about 5 minutes to 120 minutes.

[0033] Enzymes that are capable of hydrolyzing proteins are commonlyreferred to as carbonyl hydrolases. In addition to hydrolyzing peptidebonds of proteins, carbonyl hydrolases, depending upon the conditions,are often capable of hydrolyzing peptide bonds of peptides, ester bondsof fatty acids, and ester bonds of triglycerides. As used herein, aprotein generally consists of at least about ten individual amino acids,whereas a peptide, which is a protein fragment, generally consists ofabout two to about nine individual amino acids. There are bothnaturally-occurring forms of carbonyl hydrolases and recombinant formsof carbonyl hydrolases. Some of the more important types ofnaturally-occurring carbonyl hydrolases include, for example, lipases,proteases, such as subtilisins and metalloproteases, and peptidehydrolases. Some non-exhaustive examples of peptide hydrolases includealpha-amino acylpeptide hydrolase, peptidyl-amino acid hydrolase,acylamino hydrolase, serine carboxypeptidase, metallocarboxypeptidase,thiol proteinase, carboxyl proteinase and metalloproteinase. Somenon-exhaustive exemplary classes of proteases may further include thiol,acid, endo, and exo proteases.

[0034] A recombinant carbonyl hydrolase is a carbonyl hydrolase that isnot naturally-occurring. A naturally-occurring carbonyl hydrolase isencoded with a naturally-occurring DNA sequence. In a recombinantcarbonyl hydrolase, the DNA sequence that would ordinarily encode thecarbonyl hydrolase has been modified into a mutant, ornon-naturally-occurring, DNA sequence. The mutant DNA sequence encodes asubstitution, insertion, and/or deletion of one or more amino acids inthe amino acid sequence that would ordinarily be present in thenaturally-occurring carbonyl hydrolase. Thus, the presence of the mutantDNA sequence, namely an amino acid sequence not found in nature, causesthe carbonyl hydrolase that includes the DNA sequence to be anon-naturally-occurring, or recombinant, carbonyl hydrolase. Theprecursor carbonyl hydrolase of any particular recombinant carbonylhydrolase may itself be either a naturally-occurring carbonyl hydrolaseor a recombinant carbonyl hydrolase. Suitable methods for modifying theamino acid sequence to yield a recombinant carbonyl hydrolase aredisclosed in U.S. Pat. Nos. 5,185,258; 5,204,015; 5,700,676; 5,763,257;5,801,038; and 5,955,350 and in PCT Publication Nos. WO 95/10615 and WO99/20771.

[0035] Enzymes that are capable of hydrolyzing proteins may also beknown as proteases, and enzymes that are capable of hydrolyzing peptidesmay also be known as peptide hydrolases. Proteases may also be referredto as proteolytic enzymes. Proteases are a form of carbonyl hydrolasesthat, under suitable conditions, may cleave peptide bonds of proteins,whereas peptide hydrolases are a form of carbonyl hydrolases that undersuitable conditions, may cleave peptide bonds of peptides. Someproteases, under suitable conditions, may also cleave peptide bonds ofpeptides, and thus may also be characterized as peptide hydrolases.Therefore, a peptide hydrolase may also be a protease. On the otherhand, a protease is not necessarily a peptide hydrolase, though someproteases are in fact peptide hydrolases.

[0036] Proteases, like carbonyl hydrolases, may be eithernaturally-occurring proteases with naturally-occurring DNA sequences orrecombinant proteases with mutant DNA sequences. Likewise, peptidehydrolases, like carbonyl hydrolases, may be either naturally-occurringpeptide hydrolases with naturally-occurring DNA sequences or recombinanthydrolases with mutant DNA sequences.

[0037] Recombinant proteases and recombinant peptide hydrolases may bedirectly derived from naturally-occurring proteases andnaturally-occurring peptide hydrolases), respectively (i.e.: when therecombinant protease or recombinant peptide hydrolase is a mutant of thenaturally-occurring protease or the naturally-occurring peptidehydrolase, respectively). Also, recombinant proteases and recombinantpeptide hydrolases may be indirectly derived from naturally-occurringproteases and naturally-occurring peptide hydrolases), respectively(where the recombinant protease or recombinant peptide hydrolase is asecond order relative of the naturally-occurring protease or thenaturally-occurring peptide hydrolase, respectively, i.e.: when a firstrecombinant protease or first recombinant peptide hydrolase is a mutantof a second recombinant protease or a second recombinant peptidehydrolase, respectively, and the second recombinant protease or thesecond recombinant peptide hydrolase is a mutant of thenaturally-occurring protease or the naturally-occurring peptidehydrolase, respectively).

[0038] Naturally-occurring proteases (and naturally-occurring peptidehydrolases) are available from many sources, including animal,vegetable, and microbial matter. Recombinant proteases and recombinantpeptide hydrolases may be directly or indirectly derived fromnaturally-occurring proteases and naturally-occurring peptidehydrolases, respectively, with any source, such as an animal, vegetable,or microbial source. Naturally-occurring proteases from any source, suchas an animal, vegetable, or microbial source, maybe employed in theprocess of the present invention, and recombinant proteases that aredirectly or indirectly derived from naturally-occurring proteases andnaturally-occurring peptide hydrolases, respectively, from any source,such as an animal, vegetable, or microbial source, may be employed inthe process of the present invention.

[0039] Trypsin and chymotrypsin, which are each pancreatic proteases,are some non-exhaustive examples of suitable naturally-occurringproteases from animal matter that may be employed in the process of thepresent invention. Ficin, bromelain, and papain are some non-exhaustiveexamples of suitable naturally-occurring proteases from vegetable matterthat may be employed in the process of the present invention. Bacillusspp., i.e.: Bacillus licheniformis, Bacillus subtilis, Bacillusalkalophilus, Bacillus cereus, Bacillus natto, and Bacillus vulgatus,which are each bacterial proteases, and Aspergillus spp., Mucor spp.,and Rhizopus spp., which are each examples of fungal proteases, are somenon-exhaustive examples of suitable naturally-occurring microbialproteases that may be employed in the process of the present invention.

[0040] A serine protease is a protease that includes a catalytic triadof three particular amino acids, namely aspartate, histidine, andserine. Like the more general protease classification, some serineproteases, under appropriate conditions, act as peptide hydrolases thatcleave peptide linkages of peptides and are consequently also properlyclassified as serine peptide hydrolases. Both naturally-occurring serineproteases and recombinant serine proteases may be employed in theprocess of the present invention. Preferably, any naturally-occurringserine proteases and any recombinant serine proteases that are employedin the process of the present invention also act as serine peptidehydrolases under the conditions employed in the process of the presentinvention.

[0041] A couple of exemplary serine proteases are subtilisins andchymotrypsins. Subtilisins are microbial proteases, and, morespecifically, have both fungal and bacterial origins. On the other hand,chymotrypsins are pancreatic enzymes with an animal origin. In thesubtilisins, the relative order of the catalytic triad of amino acids(aspartate, histidine, and serine), reading from the amino to carboxyterminus of the triad, is aspartate-histidine-serine. In thechymotrypsins, the relative order of the catalytic triad of amino acids(aspartate, histidine, and serine), reading from the amino to carboxyterminus of the triad, is, however, histidine-aspartate-serine. Thus, asubtilisin is a serine protease that has the catalytic triad of aminoacids arranged in the aspartate-histidine-serine order.Naturally-occurring or recombinant subtilisins may be employed in theprocess of the present invention. Preferably, any naturally-occurring orrecombinant subtilisin that is employed in the process of the presentinvention also acts as a peptide hydrolase under the conditions employedin the process of the present invention.

[0042] Bacillus subtilisins are subtilisin proteases with a microbialorigin. Like the more general protease classification, some bacillussubtilisins, under appropriate conditions, act as peptide hydrolasesthat cleave peptide linkages of peptides and are consequently alsoproperly characterized as bacillus subtilisin peptide hydrolases. Bothnaturally-occurring bacillus subtilisins and recombinant bacillussubtilisins may be employed in the process of the present invention.Preferably, any naturally-occurring bacillus subtilisins and anyrecombinant bacillus subtilisins that are employed in the process of thepresent invention also act as bacillus subtilisin peptide hydrolasesunder the conditions employed in the process of the present invention.

[0043] A series of naturally-occurring bacillus subtilisins is known tobe produced and secreted by various microbial species, such as B.amyloliquefaciens, B. licheniformis, B. subtilis, and B. pumilus, forexample. Though the amino acid sequences of the members of thisnaturally-occurring bacillus subtilisin series are not entirelyhomologous, the subtilisins in this series tend to exhibit the same orsimilar type of proteolytic activity, though stability issues do existfor some members of this series. Also, conditions for satisfactoryactivity levels vary somewhat between some members of this series.Furthermore, it is believed that some members of this series exhibitstrong peptide hydrolase activity, whereas other members of this seriesexhibit little if any peptide hydrolase activity. The exemplary bacillussubtilisins provided above may be divided into two groups: (1) thesubtilisins secreted by B. licheniformis (subtilisin Carlsberg) and B.pumilus, which are generally less stable below a pH of about 9.0 and (2)the subtilisins secreted by B. amyloliquefaciens (subtilisin Novo;subtilisin BPN) and by B. subtilis. Both naturally-occurring subtilisinssecreted by B. licheniformis, B. amyloliquefaciens, and B. subtilis, aswell as, recombinant subtilisins that are directly or indirectly derivedfrom any of these naturally-occurring subtilisins may be employed in theprocess of the present invention.

[0044] In one preferred form, a recombinant subtilisin that is obtainedthrough recombinant means is employed as the proteolytic enzyme in theprocess of the present invention. As used herein, the term “recombinantsubtilisin” refers to a subtilisin in which the DNA sequence encodingthe subtilisin is modified to produce a mutant DNA sequence that encodesthe substitution, deletion, and/or insertion of one or more amino acidsin the naturally-occurring subtilisin amino acid sequence that wouldotherwise exist. As one non-exhaustive example, the recombinantsubtilisin may have methionine substituted at amino acid residues 50,124, and 222 in place of phenylalanine, isoleucine, and glutamine,respectively.

[0045] Recombinant methods to obtain genes that encode eithernaturally-occurring precursor subtilisins or recombinant precursorsubtilisins are known in the art. The methods generally entailsynthesizing labeled probes with putative sequences that encode regionsof the protease of interest, preparing genomic libraries from organismsexpressing the protease of interest, and screening the libraries for thegene of interest by hybridization to the labeled probes. Positivelyhybridizing clones are then mapped and sequenced.

[0046] The identified protease gene is then ligated into a high copynumber plasmid. The high copy number plasmid with the ligated proteasegene is then used to transform a host cell and express the protease ofinterest. This plasmid replicates in hosts in the sense that the plasmidcontains the well-known elements necessary for plasmid replication: (1)a promoter operably linked to the gene of interest (which may besupplied as the gene's own homologous promoter if the promoter isrecognized, i.e., transcribed by the host), (2) a transcriptiontermination and polyadenylation region (necessary for stability of themRNA transcribed by the host from the protease gene in certaineucaryotic host cells) that is exogenous or is supplied by theendogenous terminator region of the protease gene, and, desirably, (3) aselection gene, such as an antibiotic resistance gene, that enablescontinuous cultural maintenance of plasmid-infected host cells by growthin antibiotic-containing media. High copy number plasmids also containan origin of replication for the host that thereby enables large numbersof plasmids to be generated in the cytoplasm without chromosomallimitations. However, it is within the scope of the present invention tointegrate multiple copies of the protease gene into a host genome. Thisis facilitated by procaryotic and eucaryotic organisms that areparticularly susceptible to homologous recombination.

[0047] The following cassette mutagenesis method may also be used tofacilitate construction of subtilisin variants (recombinant forms ofsubtilisin) that may be employed in the process of the presentinvention, although other methods known to those of ordinary skill inthe art may also be used. First, the naturally-occurring gene encodingthe subtilisin is obtained and sequenced in whole or in part. Then, thesequence is scanned for a point at which mutation (deletion, insertion,and/or substitution) of one or more amino acids in the encoded enzyme isdesired. The amino acid sequences flanking this desired mutation pointare evaluated for the presence of restriction sites that supportreplacement of a short segment of the gene with an oligonucleotide poolthat, when expressed, will encode various mutants. Such restrictionsites are preferably unique sites within the protease gene to facilitatereplacement of the gene segment. However, any convenient restrictionsite that is not overly redundant in the protease gene may be used,provided the gene fragments generated by restriction digestion may bereassembled in proper sequence. If restriction sites are not present atlocations within a convenient distance from the desired mutation point(from 10 to 15 nucleotides), suitable restriction sites are generated bysubstituting nucleotides in the gene without causing a change in eitherthe reading frame or the amino acids that are encoded in the finalconstruction.

[0048] Mutation of the gene to change the sequence of the gene andconform to the desired sequence is accomplished by M13 primer extensionin accordance with generally known methods. The task of locatingsuitable flanking regions and evaluating the needed changes to arrive attwo convenient restriction site sequences is made routine by theredundancy of the genetic code, a restriction enzyme map of the gene,and the large number of different restriction enzymes. Note that if aconvenient flanking restriction site is available, the above method needbe used only in connection with the flanking region that does notcontain a site.

[0049] The gene may be naturally-occurring gene, a variant of anaturally-occurring gene, or a synthetic gene. A synthetic gene encodinga naturally-occurring or mutant precursor subtilisin may be produced bydetermining the DNA and/or amino acid sequence of a precursorsubtilisin. Multiple, overlapping, synthetic single-stranded DNAfragments are thereafter synthesized, which upon hybridization andligation produce a synthetic DNA encoding the precursor protease. Anexample of a synthetic gene construction is set forth in Example 3 ofU.S. Pat. No. 5,204,015. The entire disclosure of U.S. Pat. No.5,204,015 is therefore incorporated herein by reference.

[0050] As one non-exhaustive example, a bacillus subtilisin such as B.amyloliquefaciens subtilisin, which is an alkaline bacterial protease,may be mutated by modifying the DNA encoding the B. amyloliquefacienssubtilisin to encode the substitution of one or more amino acids ofvarious amino acid residues within the mature form of the recombinantsubtilisin product. These mutant subtilisins have at least one propertythat is different when compared to the same property of the precursorsubtilisin. Properties that may be modified fall into severalcategories: oxidative stability, substrate specificity, thermalstability, alkaline stability, catalytic activity, pH activity profile,resistance to proteolytic degradation, K_(m), kcat and K_(m) over kcatratio.

[0051] Though extended discussion is provided herein about alkalineproteases that may be derived from B. amyloliquefaciens, it is to beunderstood that any other alkaline protease, such as alkaline proteasesof Aspergillus sp., Dendryphiella sp., Scolebasidium sp., Candidalipolytica, Yarrowia lipolytica, Aureobasidium pullulans; Streptomycessp., like Strepomyces rectus var. proteolyticus NRRL 3150, Streptomycessp. YSA-130, S. diastaticus SS1, S. corchorusii ST36, S. pactum DSM40530; alkalophilic actinomycetes, such as Nocardiopsis dassonvillei,and Oerskovia xanthineolytica TK-1; Pseudomonas aeruginosa, Pseudomonasmaltophila, or Pseudomonas sp. Strain B45; Xanthomonas maltophil; Vibrioalginolyticus, or Vibrio metschnikovii strain RH530; Kurthia spiroforme;Psiloteredo healdi; Halophiles, such as Halobacterium sp., likeHalobacterium halobium ATCC 43214, or Halomonas sp. ES-10, may beemployed in the process of the present invention to realize benefits ofthe present invention.

[0052] The alkaline proteolytic enzyme that is employed in the processof the present invention is preferably a bacterial alkaline proteolyticenzyme. More preferably, the bacterial alkaline proteolytic enzyme isderived from a genetically modified strain of bacteria belonging to thespecies subtilis of the genus Bacillus. Still more preferably, thebacterial alkaline proteolytic enzyme belongs to the speciesamyloliquefaciens of the genus Bacillus. Even more preferably, thebacterial alkaline proteolytic enzyme belongs to the speciesamyloliquefaciens of the genus Bacillus that is expressed by agenetically modified strain of bacteria belonging to the speciessubtilis of the genus Bacillus. As one suitable example, the alkalineproteolytic enzyme may be the alkaline proteolytic enzyme present in theMULTIFECT® P-3000 enzyme composition that is available from GenencorInternational, Inc. of Santa Clara, Calif.

[0053] The enzyme of the MULTIFECT® P-3000 enzyme composition is abacterial alkaline proteolytic enzyme that belongs to the speciesamyloliquefaciens of the genus Bacillus and is expressed by agenetically-modified strain of bacteria belonging to the speciessubtilis of the genus Bacillus. The enzyme of the MULTIFECT® P-3000enzyme composition is commonly known as a subtilisin. The MULTIFECT®P-3000 enzyme composition includes the bacterial alkaline proteolyticenzyme, along with a carrier (propylene glycol) that is compatible withthe bacterial alkaline proteolytic enzyme. The MULTIFECT® P-3000 enzymecomposition may be combined with the slurried vegetable protein feed atany concentration that is effective to modify the proteinaceouscomponents of the slurried vegetable protein feed in accordance with thepresent invention. As one non-exhaustive example, the MULTIFECT® P-3000enzyme composition may be combined with the slurried vegetable proteinfeed at a ratio ranging from about ½ pound (about 227 grams) of theMULTIFECT® P-3000 enzyme composition per 100 pounds (45.35 kilograms) ofvegetable protein material to about two pounds (about 907 grams) of theMULTIFECT® P-3000 enzyme composition per 100 pounds (45.35 kilograms) ofvegetable protein material.

[0054] Though extended discussion is provided herein about proteasesthat may be derived from specific sources, it is to be understood thatproteases, generally, such as naturally-occurring proteases from anysource (including, for example, an animal, vegetable, or microbialsource) may be employed in the process of the present invention, andrecombinant proteases that are directly or indirectly derived fromnaturally-occurring proteases and naturally-occurring peptidehydrolases, respectively, from any source (including, for example, ananimal, vegetable, or microbial source) may be employed in the processof the present invention. Also, naturally-occurring serine proteases andrecombinant serine proteases may be employed in the process of thepresent invention.

[0055] Additionally, naturally-occurring or recombinant subtilisinsmaybe employed in the process of the present invention.

[0056] Furthermore, both naturally-occurring bacillus subtilisins andrecombinant bacillus subtilisins maybe employed in the process of thepresent invention. Likewise, both naturally-occurring subtilisinssecreted by B. licheniformis, B. amyloliquefaciens, and B. subtilis, aswell as, recombinant subtilisins that are directly or indirectly derivedfrom any of these naturally-occurring subtilisins may be employed in theprocess of the present invention.

[0057] Preferably, any naturally-occurring proteases and any recombinantproteases that are employed in the process of the present invention, nomatter the source or derivation of the naturally-occurring proteases andany recombinant proteases, also act as peptide hydrolases under theconditions employed in the process of the present invention.

[0058] As used herein, proteolytic activity is defined as the rate ofhydrolysis of peptide bonds per milligram of active enzyme. Many wellknown procedures exist for measuring proteolytic activity (K. M. Kalisz,“Microbial Proteinases,” Advances in BiochemicalEngineering/Biotechnology, A. Fiechter ed., 1988). Techniques todetermine such activities are well known in the art and may be used inthe present invention for determining an appropriate concentration ofprotease to be employed in the process of the present invention.

[0059] Determining the optimum conditions for operation of a proteaseare routine for a worker of ordinary skill in the art. Through routinemethods, it is possible to determine the working pH range, the optimumpH, the working temperature range, the optimum temperature range and thepresence of cofactors and enzyme activators necessary to obtain suitableperformance from the protease for the given task. In general, if acertain set of conditions are necessary for a particular application, itis possible to select a protease which has optimal activity under thoseconditions. Subtilisins are generally active in the alkaline range,i.e., at pHs greater than about 7 standard pH units, and at temperaturesfrom about 10° C. to about 80° C.

[0060] The alkaline proteolytic enzyme(s) incorporated in the process ofthe present invention may be characterized as a protease that exhibitsproteolytic activity at alkaline pHs, such as at a pH of about 7standard pH units, or more. The specific level of activity of thealkaline proteolytic enzyme(s) should be effective to modify theproteinaceous components of the slurried vegetable protein feed inaccordance with the present invention. Consequently, the process of thepresent invention is not limited to any particular level of activity ofthe alkaline proteolytic enzyme(s).

[0061] Following enzymatic hydrolysis of the slurried vegetable proteinfeed to form the slurried vegetable protein product, the proteolyticenzyme, such as the preferred alkaline proteolytic enzyme, isdeactivated by heating the slurried vegetable protein product to atemperature of at least about 85° C., or more, for a period of at leastabout one to about two minutes, or more, preferably for a period ofabout 5 minutes, or more, and more preferably for a period of about 5minutes to about 10 minutes. Temperatures at or above about 85° C. areusually sufficient to inactivate the proteolytic enzyme, such as thepreferred alkaline proteolytic enzyme.

[0062] Beyond the objective of inactivating the proteolytic enzyme, theheating step and the manner in which the heating step is performed arenot believed to be critical to achieving the benefits of the presentinvention. Furthermore, the heating step may be achieved by heating theslurried vegetable protein product in the batch reactor or bycirculating the slurried vegetable protein product through a heatexchanger, a jet cooker, or any similar heating device of the typetypically employed for heating food products in the food manufacturingindustry.

[0063] Following inactivation of the proteolytic enzyme, such as thepreferred alkaline proteolytic enzyme, the slurried vegetable proteinproduct may be comminuted to ensure that any fibrous material is brokenapart prior to drying the slurried vegetable protein product.Alternatively, the comminution may be carried out prior to inactivatingthe proteolytic enzyme. In any event, comminution ensures uniformity ofthe slurried vegetable protein product and helps to ensure that uniformdrying occurs. One example of suitable equipment for achieving adequatecomminution is the COMITROL® Model No. 1700 processor that is availablefrom Urschel Laboratories, Inc of Valparaiso, Ind.

[0064] Following proteolytic enzyme inactivation and any comminution,the slurried vegetable protein product is dried. The slurried vegetableprotein product may be dried using any drying technique or equipment,such as a drum dryer, a vibrating bed dryer, or any type of flash dryer.However, the slurried vegetable protein product is preferably flashdried because flash drying creates a uniform powdered product. Spraydrying is the most commonly used flash drying technique, though freezedrying may also be employed. Some examples of suitable spray dryersinclude vertical spray dryers (VRS dryers) and horizontal spray dryers(HRS dryers) that are available from C. E. Rogers Co. of Northville,Mich., and tower spray dryers that are available from Niro Inc. ofColumbia, Md. The slurried vegetable protein product may optionally becooled, such as to a temperature of about 65° C., prior to drying. Thedrying step transforms the slurried vegetable protein product intopowdered vegetable protein product.

[0065] The enzymatic hydrolysis that is accomplished in accordance withthe present invention yields a number of different benefits. Forexample, the enzymatic hydrolysis dramatically decreases theconcentration of both glycinin and β-conglycinin, the predominantantigenic proteins, in the powdered vegetable protein product ascompared to the concentration of these antigenic proteins in thevegetable protein material that is used to form the slurried vegetableprotein feed. This reduction of antigenic protein content in thepowdered vegetable protein product greatly reduces the likelihood thatuse of the powdered vegetable protein product in animal feed and humanfood would lead to the development of allergies and/or difficultiesdigesting the powdered vegetable protein product.

[0066] As used herein, unless otherwise indicated, the concentration ofglycinin in the vegetable protein material is expressed in terms of theweight of glycinin in the vegetable protein material relative to theweight of crude protein in the vegetable protein material, theconcentration of glycinin in the slurried vegetable protein product isexpressed in terms of the weight of glycinin in the slurried vegetableprotein product relative to the weight of crude protein in the slurriedvegetable protein product, and the concentration of glycinin in thepowdered vegetable protein product is expressed in terms of the weightof glycinin in the powdered vegetable protein product relative to theweight of crude protein in the powdered vegetable protein product. Also,as used herein, unless otherwise indicated, the concentration ofβ-conglycinin in the vegetable protein material is expressed in terms ofthe weight of β-conglycinin in the vegetable protein material, theconcentration of β-conglycinin in the slurried vegetable protein productis expressed in terms of the weight of β-conglycinin in the slurriedvegetable protein product relative to the weight of crude protein in theslurried vegetable protein product, and the concentration ofβ-conglycinin in the powdered vegetable protein product is expressed interms of the weight of β-conglycinin in the powdered vegetable proteinproduct relative to the weight of crude protein in the powderedvegetable protein product.

[0067] The particular proteolytic enzyme(s) employed in the enzymatichydrolysis of the present invention, such as the preferred alkalineproteolytic enzyme(s), in combination with the conditions present duringthe enzymatic hydrolysis and the enzyme deactivation step of the presentinvention, should be effective (1) to reduce the concentration ofglycinin by at least about 50 percent, more preferably by at least about70 percent, and most preferably by at least about 85 percent, in thepowdered vegetable protein product as compared to the vegetable proteinmaterial and (2) to reduce the concentration of β-conglycinin by atleast 99 percent, more preferably by about 100 percent, and mostpreferably by 100 percent, in the powdered vegetable protein product ascompared to the concentration of β-conglycinin in the vegetable proteinmaterial. Furthermore, the particular proteolytic enzyme(s), such as thepreferred alkaline proteolytic enzyme(s), and the conditions employedduring the enzymatic hydrolysis and the enzyme deactivation step shouldbe effective to reduce the combined concentration of glycinin andβ-conglycinin by at least about 70 percent, more preferably by at leastabout 80 percent, and most preferably by at least about 92 percent inthe powdered vegetable protein product, as compared to the combinedconcentration of glycinin and β-conglycinin in the vegetable proteinmaterial.

[0068] When (1) the slurried vegetable feed has a pH of at least about7.0 standard pH units, preferably at least about 8.5 standard pH units,more preferably above about 8.5 standard pH units to about 9.5 standardpH units, and even more preferably from about 9.0 standard pH units toabout 9.5 standard pH units and (2) the period of enzymatic hydrolysisis about 5 minutes to about 120 minutes, preferably about 5 to about 90minutes, and more preferably about 5 to about 60 minutes, the particularproteolytic enzyme employed in the enzymatic hydrolysis of the presentinvention, in combination with the conditions present during theenzymatic hydrolysis period (including, but not limited to, the pHconditions and time of enzymatic hydrolysis that are referred to in (1)and (2) above) and the enzyme deactivation step of the presentinvention, is preferably effective (a) to reduce the concentration ofglycinin by at least about 50 percent, more preferably by at least about70 percent, and most preferably by at least about 85 percent, in thepowdered vegetable protein product as compared to the vegetable proteinmaterial and/or (b) to reduce the concentration of β-conglycinin by atleast 99 percent, more preferably by about 100 percent, and mostpreferably by 100 percent, in the powdered vegetable protein product ascompared to the concentration of β-conglycinin in the vegetable proteinmaterial.

[0069] When (1) the slurried vegetable feed has a pH of at least about7.0 standard pH units, preferably at least about 8.5 standard pH units,more preferably above about 8.5 standard pH units to about 9.5 standardpH units, and even more preferably from about 9.0 standard pH units toabout 9.5 standard pH units and (2) the period of enzymatic hydrolysisis about 5 minutes to about 120 minutes, preferably about 5 to about 90minutes, and more preferably about 5 to about 60 minutes, the particularproteolytic enzyme employed in the enzymatic hydrolysis of the presentinvention, in combination with the conditions present during theenzymatic hydrolysis period (including, but not limited to, the pHconditions and time of enzymatic hydrolysis that are referred to in (1)and (2) above) and the enzyme deactivation step of the presentinvention, is preferably effective to reduce the combined concentrationof glycinin and β-conglycinin by at least about 70 percent, morepreferably by at least about 80 percent, and most preferably by at leastabout 92 percent in the powdered vegetable protein product, as comparedto the combined concentration of glycinin and P-conglycinin in thevegetable protein material.

[0070] Another benefit of the process of the present invention is theenhanced solubility of the powdered vegetable protein product in water,as compared to the solubility of the vegetable protein material inwater. Besides reducing the antigenicity of the powdered vegetableprotein product, the process of the present invention additionallyenhances the water solubility of proteins present in the powderedvegetable protein product, as compared to the water solubility of theproteins present in the vegetable protein material. The solubility ofprotein in a particular sample may be characterized based upon theProtein Dispersion Index (PDI) of the sample.

[0071] When the vegetable protein material has a PDI of about 60percent, or more, the process of the present invention is effective toincrease the PDI of the powdered vegetable protein product, as comparedto the PDI of the vegetable protein material, by at least about 20percent (for example, changing from a starting PDI of about 62 percentto a PDI of at least about 82 percent), more preferably by at leastabout 23 percent (for example, changing from a starting PDI of about 62percent to a PDI of at least about 85 percent), and most preferably byat least about 26 percent (for example, changing from a starting PDI ofabout 62 percent to a PDI of at least about 88 percent). When the PDI ofthe vegetable protein material is less than about 60 percent, theprocess of the present invention is effective to increase the PDI of thepowdered vegetable protein product that is based upon the vegetableprotein material to greater than about 60 percent, is preferablyeffective to increase the PDI of the powdered vegetable protein productto at least about 70 percent, and is more preferably effective toincrease the PDI of the powdered vegetable protein product to at leastabout 80 percent.

[0072] Preferably, when the PDI of the vegetable protein material isabout 40 percent, or less, the process of the present invention iseffective to increase the PDI of the powdered vegetable protein productthat is based upon the vegetable protein material to greater than about60 percent, is more preferably effective to increase the PDI of thepowdered vegetable protein product to at least about 70 percent, and isstill more preferably effective to increase the PDI of the powderedvegetable protein product to at least about 80 percent. More preferably,when the PDI of the vegetable protein material is about 20 percent, orless, the process of the present invention is effective to increase thePDI of the powdered vegetable protein product that is based upon thevegetable protein material to greater than about 60 percent, is stillmore preferably effective to increase the PDI of the powdered vegetableprotein product to at least about 70 percent, and is even morepreferably effective to increase the PDI of the powdered vegetableprotein product to at least about 80 percent.

[0073] When (1) the slurried vegetable feed has a pH of at least about7.0 standard pH units, preferably at least about 8.5 standard pH units,more preferably above about 8.5 standard pH units to about 9.5 standardpH units, and even more preferably from about 9.0 standard pH units toabout 9.5 standard pH units, (2) the period of enzymatic hydrolysis isabout 5 minutes to about 120 minutes, preferably about 5 to about 90minutes, and more preferably about 5 to about 60 minutes, and (3) thevegetable protein material has a PDI of about 60 percent, or more, theparticular proteolytic enzyme employed in the enzymatic hydrolysis ofthe present invention, in combination with the conditions present duringthe enzymatic hydrolysis period (including, but not limited to, the pHconditions and time of enzymatic hydrolysis that are referred to in (1)and (2) above) and the enzyme deactivation step of the presentinvention, are preferably effective to increase the PDI of the powderedvegetable protein product, as compared to the PDI of the vegetableprotein material, by at least about 20 percent, more preferably by atleast about 23 percent, and most preferably by at least about 26percent.

[0074] When (1) the slurried vegetable feed has a pH of at least about7.0 standard pH units, preferably at least about 8.5 standard pH units,more preferably above about 8.5 standard pH units to about 9.5 standardpH units, and even more preferably from about 9.0 standard pH units toabout 9.5 standard pH units, (2) the period of enzymatic hydrolysis isabout 5 minutes to about 120 minutes, preferably about 5 to about 90minutes, and more preferably about 5 to about 60 minutes, and thevegetable protein material has a PDI of less than about 60 percent, theparticular proteolytic enzyme employed in the enzymatic hydrolysis ofthe present invention, in combination with the conditions present duringthe enzymatic hydrolysis period (including, but not limited to, the pHconditions and time of enzymatic hydrolysis that are referred to in (1)and (2) above) and the enzyme deactivation step of the presentinvention, are preferably effective to increase the PDI of the powderedvegetable protein product that is based upon the vegetable proteinmaterial to greater than about 60 percent, more preferably to at leastabout 70 percent, and still more preferably to at least about 80percent.

[0075] When (1) the slurried vegetable feed has a pH of at least about7.0 standard pH units, preferably at least about 8.5 standard pH units,more preferably above about 8.5 standard pH units to about 9.5 standardpH units, and even more preferably from about 9.0 standard pH units toabout 9.5 standard pH units, (2) the period of enzymatic hydrolysis isabout 5 minutes to about 120 minutes, preferably about 5 to about 90minutes, and more preferably about 5 to about 60 minutes, and thevegetable protein material has a PDI of about 40 percent, or less, theparticular proteolytic enzyme employed in the enzymatic hydrolysis ofthe present invention, in combination with the conditions present duringthe enzymatic hydrolysis period (including, but not limited to, the pHconditions and time of enzymatic hydrolysis that are referred to in (1)and (2) above) and the enzyme deactivation step of the presentinvention, are preferably effective to increase the PDI of the powderedvegetable protein product that is based upon the vegetable proteinmaterial to greater than about 60 percent, more preferably to at leastabout 70 percent, and still more preferably to at least about 80percent.

[0076] When (1) the slurried vegetable feed has a pH of at least about7.0 standard pH units, preferably at least about 8.5 standard pH units,more preferably above about 8.5 standard pH units to about 9.5 standardpH units, and even more preferably from about 9.0 standard pH units toabout 9.5 standard pH units, (2) the period of enzymatic hydrolysis isabout 5 minutes to about 120 minutes, preferably about 5 to about 90minutes, and more preferably about 5 to about 60 minutes, and thevegetable protein material has a PDI of about 20 percent, or less, theparticular proteolytic enzyme employed in the enzymatic hydrolysis ofthe present invention, in combination with the conditions present duringthe enzymatic hydrolysis period (including, but not limited to, the pHconditions and time of enzymatic hydrolysis that are referred to in (1)and (2) above) and the enzyme deactivation step of the presentinvention, are preferably effective to increase the PDI of the powderedvegetable protein product that is based upon the vegetable proteinmaterial to greater than about 60 percent, more preferably to at leastabout 70 percent, and still more preferably to at least about 80percent.

[0077] The enhanced solubility of the powdered vegetable protein productin water, as compared to the solubility of the vegetable proteinmaterial in water, is believed to be due in significant part to theprotein (or peptide) molecular weight reduction that is achieved in thepowdered vegetable protein product, as compared to the protein molecularweight of the vegetable protein material. In this regard, when thevegetable protein material has an average protein molecular weight inthe range of about 125 kilodaltons to about 440 kilodaltons, the processof the present invention is preferably effective to produce the powderedvegetable protein product with an average protein molecular weight ofabout 7500 Daltons or less, more preferably about 5000 Daltons or less,still more preferably about 2500 Daltons or less, even more preferablyabout 2000 Daltons or less, yet more preferably about 1500 Daltons orless, and most preferably about 1250 Daltons or less. As used herein,the term “average protein molecular weight” means the average molecularweight of both proteins and protein fragments (peptides) in the samplebeing considered.

[0078] When (1) the slurried vegetable feed has a pH of at least about7.0 standard pH units, preferably at least about 8.5 standard pH units,more preferably above about 8.5 standard pH units to about 9.5 standardpH units, and even more preferably from about 9.0 standard pH units toabout 9.5 standard pH units, (2) the period of enzymatic hydrolysis isabout 5 minutes to about 120 minutes, preferably about 5 to about 90minutes, and more preferably about 5 to about 60 minutes, and (3) thevegetable protein material has an average protein molecular weight inthe range of about 125 kilodaltons to about 440 kilodaltons, theparticular proteolytic enzyme employed in the enzymatic hydrolysis ofthe present invention, in combination with the conditions present duringthe enzymatic hydrolysis period (including, but not limited to, the pHconditions and time of enzymatic hydrolysis that are referred to in (1)and (2) above) and the enzyme deactivation step of the presentinvention, are preferably effective to produce the powdered vegetableprotein product with an average protein molecular weight of about 7500Daltons or less, more preferably about 5000 Daltons or less, still morepreferably about 2500 Daltons or less, even more preferably about 2000Daltons or less, yet more preferably about 1500 Daltons or less, andmost preferably about 1250 Daltons or less.

[0079] Furthermore, beyond reducing antigenicity levels in the powderedvegetable protein product and increasing protein solubility in thepowdered vegetable protein product, as compared to antigenicity levelsand protein solubility in the vegetable protein material, the process ofthe present invention additionally tends to reduce off-flavors in thepowdered vegetable protein product, as compared to off-flavors presentin the vegetable protein material.

[0080] Thus, three major beneficial aspects of the process of thepresent invention include reducing antigenicity levels in the powderedvegetable protein product, increasing protein solubility in the powderedvegetable protein product, and reducing off-flavors in the powderedvegetable protein product, as compared to the levels of these variablesin the vegetable protein material. Consequently, after drying, thepowdered vegetable protein product may be employed in a wide variety offood substrates, destined for consumption by both animals and humans, toincrease the nutritional value of the food substrates. For example, thepowdered vegetable protein product may be incorporated in milk replacersfor feeding monogastric mammals, such as human babies and young animalswith only one functioning stomach, such as young calves, while enhancingthe solubility and stability of the powdered vegetable protein productin the milk replacer and reducing chances for allergic reaction in themammals fed the milk replacer. Furthermore, the powdered vegetableprotein product may be incorporated in a number of different humanfoods, such as gelatins, beverages, and other foods that would benefitfrom a highly soluble source of protein with low propensity for allergicinducement.

Property Determination & Characterization Techniques

[0081] Various analytical techniques are employed herein. An explanationof these techniques follows. All values presented in this document forweight percent dry matter for a particular sample are based on the “asis” form of the sample and are therefore on a “wet basis,” unlessotherwise specified herein. All values presented in this document forcertain other parameters in a sample, namely, weight percent organicmatter, weight percent ash, and weight percent crude protein, are basedon the dry matter weight of the sample and are therefore on a “drymatter” or “dry” basis, unless otherwise specified herein. Furthermore,all values presented in this document for weight percent soluble proteinand for concentrations of glycinin and β-conglycinin in a particularsample are based upon the weight of crude protein in the sample, unlessotherwise specified herein.

pH Determinations

[0082] Unless otherwise indicated, all pH determinations recited orspecified herein are based upon use of the Model No. 59003-00 DigitalBenchtop pH/mV Meter that is available from Cole-Parmer Instrument Co.of Vernon Hills, Ill. using the procedure set forth in the instructionsaccompanying the Model No. 59003-00 Digital Benchtop pH/mV Meter. All pHvalues recited herein were determined at or are based upon a sampletemperature of about 25° C.

Dry Matter Weight Determination

[0083] The weight percent of dry matter in a particular sample, basedupon the total weight of the sample, is calculated after firstdetermining the moisture content in the sample. The weight of moisturein a particular sample is determined by analyzing the sample inaccordance with Method #930.15 (4.106) of Official Methods of Analysis,Association of Official Analytical Chemists (AOAC) (16^(th) Ed., 1995).The weight percent moisture in the sample, based upon the total weightof the sample, is then calculated by dividing the actual weight ofmoisture in the sample by the total weight of the sample and thenmultiplying the result of this division by 100%. The weight percent drymatter in the sample is then determined by subtracting 100% from theweight percent of moisture in the sample. For example, if a particularsample had a moisture concentration of 22 weight percent, then the drymatter content of that sample would be 78 weight percent. The weightpercent dry matter in the is also known as the weight percent totalsolids in the sample.

Ash and Organic Matter Determinations

[0084] The weight percent ash, dry basis, in a particular sample isdetermined after first determining the weight of ash in the sample. Theweight of ash in a particular sample is determined by analyzing thesample in accordance with Method #942.05 (4.1.10) of Official Methods OfAnalysis, Association of Official Analytical Chemist (AOAC) (16^(th)Ed., 1995). The weight percent ash, dry basis, in the sample is thencalculated by dividing the actual weight of ash by the weight of drymatter in the sample, that is determined by Method #930.15 as describedabove, and then multiplying this result of this division by 100%. Theweight percent organic matter, dry basis, in the sample is thencalculated by subtracting the weight percent ash, dry basis, in thesample from 100%. Thus, if the weight percent ash, dry basis, in aparticular sample is 30 weight percent, the weight percent organicmatter, dry basis, in the sample is consequently 70 weight percent.

Crude Protein Determination

[0085] The weight percent crude protein, dry basis, in a particularsample is calculated after first determining the actual weight of totalprotein in the sample. The actual weight of total protein in the sampleis determined in accordance with Method #991.20 (33.2.11) of OfficialMethods of Analysis, Association of Official Analytical Chemists (AOAC)(16^(th) Ed., 1995). The value determined by the above method yields“total Kjeldahl nitrogen,” which is equivalent to “total protein,” sincethe above method incorporates a factor that accounts for the averageamount of nitrogen in protein. Total Kjeldahl nitrogen and total proteinare sometimes referred to in the dairy industry as “crude protein.”Consequently, the terms “total Kjeldahl nitrogen,” “crude protein,” and“total protein” are used interchangeably herein. Furthermore, thoseskilled in the art will recognize that the term “total Kjeldahlnitrogen” is generally used in the art to mean “crude protein” or “totalprotein” with the understanding that the above-noted nitrogen factor hasbeen applied.

[0086] The weight percent crude protein, dry basis, in the sample iscalculated by dividing the actual weight of crude protein (a.k.a. totalKjeldahl nitrogen) by the weight of dry matter in the sample, that isdetermined by Method #930.15 as described above, and then multiplyingthis result by 100%. The weight percent crude protein in the sample,based on the organic matter content of the sample, is calculated bydividing the weight percent crude protein, dry basis, of the sample bythe weight percent organic matter, dry basis, in the sample, determinedas described above in Ash and Organic Matter Determinations, andmultiplying the result of this division by 100%.

Protein Dispersability Index (PDI) Determination

[0087] This method is used to determine the Protein Dispersability Index(PDI) of a particular sample that contains protein. The ProteinDispersability Index is a measure of the soluble protein content in asample, expressed as a percent, by weight, of the crude protein weightin the sample. Consequently, the Protein Dispersability Index isequivalent to the weight percent of soluble protein in a sample, basedupon the weight of crude protein in the sample. The ProteinDispersability Index (PDI) of a particular sample that contains proteinmay be determined in accordance with Method No. 46-24 (1995), entitledProtein Dispersability Index, of the American Association of CerealChemists (AACC). The current address of the American Association ofCereal Chemists is 3340 Pilot Knob Road, St. Paul, Minn. 55121.

Brookfield Viscosity Determination

[0088] Unless otherwise indicated, all viscosities recited herein weredetermined using a Brookfield Model No. DV-II+ viscometer that may beobtained from Brookfield Engineering Laboratories of Middleboro, Mass.Any of spindle nos. 4, 5, and/or 6 that are available from BrookfieldEngineering Laboratories for use with the Model No. DV-II+ viscometermay be used when determining the viscosity of a particular sample.Viscosity determinations were conducted in accordance with the OperatingInstructions manual for the Brookfield Model No. DV-II+ viscometer,unless otherwise indicated herein. Unless otherwise indicated herein,viscosity measurements were determined with the sample at a particulartemperature and, consequently, sample temperatures are provided witheach viscosity determination provided herein.

Protein Fragment Size Analysis By HPLC

[0089] The molecular weight distribution (or profile) for proteins andpeptides in different samples may be determined using High PressureLiquid Chromatography (“HPLC”). A Waters High Pressure LiquidChromatography system employing a Waters 510 high pressure pump, aWaters 712 WISP automatic sample injection system, and a Waters996Photodiode Array detector may be used. The Waters High PressureLiquid Chromatography system employing the specified components maybeobtained from Waters Corporation of Milford, Mass.

[0090] Some non-exhaustive examples of samples that maybe analyzed bythis HPLC method include supernatant samples obtained after centrifuginga solution of the vegetable protein feed or a solution of the powderedvegetable protein product. The solution of the vegetable protein feed orof the powdered vegetable protein product maybe prepared by blendingtogether about 3.2 grams of the vegetable protein feed or of thepowdered vegetable protein product with about 40 milliliters ofdistilled, deionized water to form a slurry. The slurry is placed in a50 milliliter centrifuge tube and then incubated at 30° C. for aboutthree hours with intermittent mixing. After the three hour incubationperiod, the 50 milliliter centrifuge tube containing the slurry isplaced in a centrifuge. After assuring that the centrifuge is balanced,the centrifuge is operated for 10 minutes at a rate of about 2700revolutions per minute. Then, the supernatant layer that forms in the 50milliliter centrifuge tube when centrifuging the slurry is used as thesample in the HPLC procedure.

[0091] In the Waters HPLC system, the Waters 996 Photodiode Arraydetector is set at 206 nanometers. The stationary phase of thechromatographic system is a BioSep SEC-S2000 size exclusion column thatmay be obtained from PHENOMENEX INC. of Torrance, Calif. The mobilephase of the chromatographic system is a solution of 100 mM sodiumphosphate with a pH of 6.8. The sample flow rate in the system is set at1.0 ml/minute for samples of the vegetable protein feed, and the sampleflow rate in the system is set at 1.0 ml/minute for samples of thepowdered vegetable protein product. The data obtained from the HPLCanalysis is printed as a graph showing molecular weight distribution(profile) of protein fragments, expressed in absorption units, as afunction of retention time. The molecular weights of proteins andpeptides in a sample, expressed in Daltons, maybe determined from astandard curve for proteins and peptides of known molecular weightanalyzed by the above-described HPLC procedure to produce a molecularweight profile for the sample. The distribution of protein molecularweights for the proteins and peptides in the sample may be averaged todetermine the average protein molecular weight of the sample.

Glycinin and β-conglycinin Determinations

[0092] The determination of Glycinin content and β-Conglycinin contentin a particular sample may be conducted in accordance with the followingprocedure, which is based upon an Enzyme-Linked Immunosorbent Assay(subsequently referred to as “ELISA”). The procedure is conducted infour separate steps: Isolation of Native Glycinin and β-Conglycinin,Antibody Preparation, ELISA Assay, and Calculations.

[0093] Isolation of Native Glycinin and β-Conglycinin

[0094] Native Glycinin and β-Conglycinin are isolated from a rawdefatted soybean flour composition by placing about three grams of theraw (i.e.: not denatured or enzymatically-degraded) defatted soybeanflour composition into fifteen milliliters (ml) of a 0.15 molar (M)sodium chloride (NaCl) solution. The mixture of the flour compositionand the NaCl solution are held for about 1 hour at 25° C., whilemaintaining the pH of the mixture at 6.7 with a 1.0 M sodium hydroxide(NaOH) solution, to form a native Glycinin and β-Conglycinin extract.The NaCl and NaOH reagents are available from Sigma Chemical Company ofSaint-Quentin Fallavier, France.

[0095] Next, the native Glycinin and β-Conglycinin extract is clarifiedby centrifugation at 1,100×g for 30 minutes at 20° C. A supernatant ofthe Glycinin and P-Conglycinin extract obtained after centrifugation isthen further purified using gel filtration. About 0.5 ml of thesupernatant is applied to a Sephacryl L S300-HR column previouslyequilibrated with a PBS buffer. The supernatant is separated into 1-mlfractions using a PBS buffer elution rate of about 100 ml per hour. ThePBS buffer should contain 0.2 grams of potassium chloride (KCl) perliter, 0.2 grams of potassium di-hydrogen phosphate (KH₂PO₄) per liter,8.0 grams of sodium chloride (NaCl) per liter, 1.14 grams of di-sodiumhydrogen phosphate (Na₂HPO₄) per liter, and 0.1 grams of Kathon perliter.

[0096] The Sephacryl L S300-HR column is available from Pharmacia ofSaint Quentin-en-Yvelines, France, while the various PBS reagents areavailable from Sigma Chemical Company of Saint-Quentin Fallavier,France. Individual purified native Glycinin and β-Conglycinin fractionsare recovered by gel filtration as single peaks at elution volumes thatcorresponded to molecular weights (MW) of 340-440 kiloDaltons (kD) forGlycinin, and 180-230 kD for β-Conglycinin. The purified native Glycininfraction and the purified native β-Conglycinin fraction are stored at−20° C. until required.

[0097] The purity of the native Glycinin fraction and the purity of thenative β-Conglycinin fraction are confirmed using sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Mini-gels (80millimeters (mm)×90 mm) include a 12.5 weight percent acrylamideseparating gel and a 4 weight percent acrylamide stacking gel. Proteinloadings are 5 microgram (μg) of protein per track for the nativeGlycinin fraction and also for the native β-Conglycinin fraction.SDS-PAGE is performed in the presence of a Laemmli buffer system thatincludes Tris-glycine containing 25 millimolar (mM) Tris, 192 mM glycineand, 2 grams of SDS per liter, at a pH of 8.3 under reducing conditionsof about 2 weight percent mercaptoethanol. Molecular weight standardsare also loaded in a separate well. Electrophoresis is performed for 1.5hours at 40 mA. Gels are stained for protein using 0.25 percentCoomassie brilliant blue R250 in methanol:acetic acid:water (5:1:4vol/vol/vol). The SDS-PAGE reagents described above are available fromSigma Chemical Company of La Verpilliere, France.

[0098] Antibody Preparation

[0099] Antisera are produced in New Zealand White Rabbits that weresupplied by Ranch Rabbits Ltd of Capthorn, Sussex. The antisera areproduced against the purified native Glycinin and β-Conglycinin obtainedin accordance with the method described above in the section of thisdocument entitled “Isolation of Glycinin and β-Conglycinin.”

[0100] Antisera for Glycinin are produced by emulsifying one (1)milligram (mg) of the purified native Glycinin in 1.0 ml of Freund'scomplete adjuvant. About 0.7 ml of this Glycinin-based emulsion isadministered intramuscularly to the rabbits on two or three occasionsover a five to seven week period. Antisera for β-Conglycinin areproduced by emulsifying one (1) milligram (mg) of the purified nativeβ-Conglycinin in 1.0 ml of Freund's complete adjuvant. About 0.7 ml ofthis P-Conglycinin-based emulsion is administered intramuscularly to therabbits on two or three occasions over a five to seven week period.

[0101] ELISA Assay

[0102] Unless otherwise indicated, all reagents used to perform theELISA Assay may be obtained from Sigma Chemical Corporation ofSaint-Quentin Fallavier, France.

[0103] 1. Sample Extraction

[0104] Soybean proteins are extracted for about 1.5 hours from a sample(also referred to herein as the “test protein sample”) of the soybeanprotein composition under consideration using 100 volumes of a boratebuffer solution at room temperature of about 22° C. The borate buffersolution has a pH of about 8.0 units and contains 100 mM SodiumPerborate (Na₂BO₃) and 0.15 M NaCl. The soybean protein extract obtainedfrom the test protein sample (also referred to herein as the “testprotein sample extract”) is clarified by centrifugation at 20,000×g for15 minutes.

[0105] 2. Glycinin Determination by ELISA Assay

[0106] a. Initial Plate Preparation

[0107] Two NUNC Immunoplate I microtitration plates, obtained from GibcoEurope, Paisley, United Kingdom, are coated with a solution containingpurified native Glycinin obtained in accordance with the methoddescribed above in the section of this document entitled “Isolation ofGlycinin and β-Conglycinin.” One of the coated plates is used fordetermining the Glycinin content of the test protein sample and one ofthe coated plates is used for determining the Glycinin content of theprotein standards samples.

[0108] Prior to coating the two plates, the purified native Glycinin isdissolved in a buffer of 50 mM sodium carbonate buffer at a pH of 9.6 toform a buffered solution containing 1 μg of purified native Glycinin perml of the buffered solution. The two plates are then coated with thepurified native Glycinin by adding 0.3 ml of the buffered solution ineach well of the plates. The two coated plates are then incubated for 16hours at 4° C. After incubation, the two coated plates are washed threetimes with a solution of TWEEN® surfactant and sodium chloride. Afterwashing, the coated and incubated plates are blotted and stored at −20°C. for no longer than 4 weeks.

[0109] b. Test Protein Sample

[0110] One of the coated and incubated plates prepared in subsection a.above entitled “Initial Plate Preparation” is employed in the ELISAassay of the Test Protein Sample. The Glycinin antisera obtained inaccordance with the method described above (see section above entitled“Antibody Preparation”) is diluted to a ratio of about 1:32,000 (v/v)with PBS. Equal volumes of the test protein sample extract (seesection 1. above entitled “Sample Extraction”) and the diluted antiseraare combined to form a mixture. Two hundred (200) μl of the mixture areadded to each well of the coated plate. The coated plate is thenincubated at 37° C. for 4 hours. After incubation, the plate is washedthree times with an aqueous solution of NaCl and TWEEN® surfactant.

[0111] After washing, 0.2 ml of anti-rabbit IgG-horseradish peroxidaseconjugate in PBS that has been diluted to 1:2000 (v/v) is added to eachwell of the coated plate. After adding the diluted anti-rabbitIgG-horseradish peroxidase conjugate, the plate is incubated for 2 hoursat 37° C. After incubation, the plate is washed three times with anaqueous solution of NaCl and TWEEN® surfactant.

[0112] After washing, aqueous solutions of2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) and hydrogenperoxide, each at a concentration of 0.01 weight percent, are added tothe coated plate, and the coated plate is incubated for 30 minutes atroom temperature. The optical density of the plate at the various wellsof the plate is then read at a detection wavelength of 405 nanometersfor 10 seconds using an Argus 300 plate reader from Packard InstrumentsCompany of Meriden, Conn.

[0113] c. Protein Standards Samples

[0114] One of the coated and incubated plates prepared in subsection a.above entitled “Initial Plate Preparation” is employed in the ELISAassay of the protein standards samples. The Glycinin antisera obtainedin accordance with the method described above (see section aboveentitled “Antibody Preparation”) is diluted to a ratio of about 1:32,000(v/v) with PBS.

[0115] A standard solution of Glycinin in PBS at a concentration of 2 mgof Glycinin per ml of the standard solution is diluted to give a rangeof different glycinin standards ranging from 100 nanograms (ng) ofglycinin per ml to 1 mg of Glycinin per ml. The number of differentglycinin standards may, as an example, be equal to the number of wellsthat are included in the plate.

[0116] For each of the individual glycinin standards, equal volumes ofthe particular glycinin standard and the diluted antisera are combinedto form a glycinin standard/antisera mixture. Therefore, the number ofglycinin standard/antisera mixtures is equal to the number of differentglycinin standards. Two hundred (200) μl of each glycininstandard/antisera mixture are added to different wells of the coatedplate. Therefore, as an example, each well of the coated plate maycontain a different one of the glycinin standard/antisera mixtures, ifthe number of different glycinin standards equals the number of wells inthe plate. The coated plate is then incubated at 37° C. for 4 hours.After incubation, the plate is washed three times with an aqueoussolution of NaCl and TWEEN® surfactant.

[0117] After washing, 0.2 ml of anti-rabbit IgG-horseradish peroxidaseconjugate in PBS that has bee diluted to 1:2000 (v/v) is added to eachwell of the coated plate. After adding the diluted anti-rabbitIgG-horseradish peroxidase conjugate, the plate is incubated for 2 hoursat 37° C. After incubation, the plate is washed three times with anaqueous solution of NaCl and TWEEN® surfactant.

[0118] After washing, aqueous solutions of2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) and hydrogenperoxide, each at a concentration of 0.01 weight percent, are added tothe coated plate, and the coated plate is incubated for 30 minutes atroom temperature. The optical density of the plate is then read at adetection wavelength of 405 nanometers for 10 seconds using an Argus 300plate reader from Packard Instruments Company of Meriden, Conn.

[0119] 3. β-Conglycinin Determination by ELISA Assay

[0120] a. Initial Plate Preparation

[0121] Two NUNC Immunoplate I microtitration plates, obtained from GibcoEurope, Paisley, United Kingdom, are coated with a solution containingpurified native β-Conglycinin obtained in accordance with the methoddescribed above in the section of this document entitled “Isolation ofGlycinin and β-Conglycinin.” One of the coated plates is used fordetermining the β-Conglycinin content of the test protein sample and oneof the coated plates is used for determining the β-Conglycinin contentof the protein standards samples.

[0122] Prior to coating the two plates, the purified nativeβ-Conglycinin is dissolved in a buffer of 50 mM sodium carbonate bufferat a pH of 9.6 to form a buffered solution containing 1 μg of purifiednative β-Conglycinin per ml of the buffered solution. The two plates arethen coated with the purified native β-Conglycinin by adding 0.3 ml ofthe buffered solution in each well of the plates. The two coated platesare then incubated for 16 hours at 4° C. After incubation, the twocoated plates are washed three times with a solution of TWEEN®surfactant and sodium chloride. After washing, the coated and incubatedplates are blotted and stored at −20° C. for no longer than 4 weeks.

[0123] b. Test Protein Sample

[0124] One of the coated and incubated plates prepared in subsection a.above entitled “Initial Plate Preparation” is employed in the ELISAassay of the Test Protein Sample. The β-Conglycinin antisera obtained inaccordance with the method described above (see section above entitled“Antibody Preparation”) is diluted to a ratio of about 1:16,000 (v/v)with PBS. Equal volumes of the test protein sample extract (seesection 1. above entitled “Sample Extraction”) and the diluted antiseraare combined to form a mixture. Two hundred (200) μl of the mixture areadded to each well of the coated plate. The coated plate is thenincubated at 37° C. for 4 hours. After incubation, the plate is washedthree times with an aqueous solution of NaCl and TWEEN® surfactant.

[0125] After washing, 0.2 ml of anti-rabbit IgG-horseradish peroxidaseconjugate in PBS that has been diluted to 1:2000 (v/v) is added to eachwell of the coated plate. After adding the diluted anti-rabbitIgG-horseradish peroxidase conjugate, the plate is incubated for 2 hoursat 37° C. After incubation, the plate is washed three times with anaqueous solution of NaCl and TWEEN® surfactant.

[0126] After washing, aqueous solutions of2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) and hydrogenperoxide, each at a concentration of 0.01 weight percent, are added tothe coated plate, and the coated plate is incubated for 30 minutes atroom temperature. The optical density of the plate at the various wellsof the plate is then read at a detection wavelength of 405 nanometersfor 10 seconds using an Argus 300 plate reader from Packard InstrumentsCompany of Meriden, Conn.

[0127] c. Protein Standards Samples

[0128] One of the coated and incubated plates prepared in subsection a.above entitled “Initial Plate Preparation” is employed in the ELISAassay of the protein standards samples. The β-Conglycinin antiseraobtained in accordance with the method described above (see sectionabove entitled “Antibody Preparation”) is diluted to a ratio of about1:16,000 (v/v) with PBS.

[0129] A standard solution of β-Conglycinin in PBS at a concentration of2 mg of β-Conglycinin per ml of the standard solution is diluted to givea range of different β-Conglycinin standards ranging from 10 nanograms(ng) of β-Conglycinin per ml to 100 μg of β-Conglycinin per ml. Thenumber of different β-Conglycinin standards may, as an example, be equalto the number of wells that are included in the plate.

[0130] For each of the individual β-Conglycinin standards, equal volumesof the particular β-Conglycinin standard and the diluted antisera arecombined to form a β-Conglycinin standard/antisera mixture. Therefore,the number of β-Conglycinin standard/antisera mixtures is equal to thenumber of different β-Conglycinin standards. Two hundred (200) μl ofeach β-Conglycinin standard/antisera mixture are added to differentwells of the coated plate. Therefore, as an example, each well of thecoated plate may contain a different one of the β-Conglycininstandard/antisera mixtures, if the number of different β-Conglycininstandards equals the number of wells in the plate. The coated plate isthen incubated at 37° C. for 4 hours. After incubation, the plate iswashed three times with an aqueous solution of NaCl and TWEEN®surfactant.

[0131] After washing, 0.2 ml of anti-rabbit IgG-horseradish peroxidaseconjugate in PBS that has bee diluted to 1:2000 (v/v) is added to eachwell of the coated plate. After adding the diluted anti-rabbitIgG-horseradish peroxidase conjugate, the plate is incubated for 2 hoursat 37° C. After incubation, the plate is washed three times with anaqueous solution of NaCl and TWEEN® surfactant.

[0132] After washing, aqueous solutions of2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) and hydrogenperoxide, each at a concentration of 0.01 weight percent, are added tothe coated plate, and the coated plate is incubated for 30 minutes atroom temperature. The optical density of the plate is then read at adetection wavelength of 405 nanometers for 10 seconds using an Argus 300plate reader from Packard Instruments Company of Meriden, Conn.

[0133] Calculations

[0134] A standard curve for glycinin content was prepared based upon theELISA analysis of the Glycinin protein standards samples. The axes ofthe standard curve included the known glycinin content of the variousGlycinin protein standards samples that were prepared and the opticaldensities measured when using the various Glycinin protein standardssamples. This standard curve for glycinin content was developed usinglinear regression after logit-log transformation. In the standard curve,the glycinin contents of the various glycinin protein standards samplesare stated relative to the crude protein content of the various glycininprotein standards samples, where crude protein contents are determinedusing the methods of Kjedahl (Crude Protein (CP)=[N]×6.25). Theconcentration of glycinin (relative to crude protein content) in testprotein sample(s) are obtained from the standard curve for glycinincontent, based upon the optical densities measured for the test proteinsample(s) when analyzing for glycinin.

[0135] Similarly, a standard curve for β-Conglycinin content wasprepared based upon the ELISA analysis of the β-Conglycinin proteinstandards samples. The axes of the standard curve included the knownβ-Conglycinin content of the various β-Conglycinin protein standardssamples that were prepared and the optical densities measured when usingthe various β-Conglycinin protein standards samples. This standard curvefor β-Conglycinin content was developed using linear regression afterlogit-log transformation. In the standard curve, the β-Conglycinincontents of the various β-Conglycinin protein standards samples arestated relative to the crude protein content of the variousβ-Conglycinin protein standards samples, where crude protein contentsare determined using the methods of Kjedahl (Crude Protein(CP)=[N]×6.25). The concentration of β-Conglycinin (relative to crudeprotein content) in test protein sample(s) are obtained from thestandard curve for β-Conglycinin content, based upon the opticaldensities measured for the test protein sample(s) when analyzing forβ-Conglycinin.

[0136] Additional Background Information About the ELISA Procedure

[0137] Additional background information about determination of Glycinincontent and β-Conglycinin content in a particular sample in accordancewith the Enzyme-Linked Immunosorbent Assay (“ELISA”) procedure that isprovided herein may be obtained from the following publications, whichare each hereby incorporated by reference herein, in their entirety:

[0138] 1. Lallès, J. P., Plumb, G. W., Mills, E. N. C., Morgan, M. R. A,Tukur, H. M., and Toullec, R., Antigenic Activity of Some SoyabeanProducts Used in Veal Calf Feeding: Comparison Between In Vitro Tests(ELISA Polyclonal vs Monoclonal) And With In Vivo Data, Pages 281-285 invan der Poel, A. F. B., Huisman, J., and Saini, H. S., ed., RecentAdvances of Research in AntiNutritional Factors in Legume Seeds, Publ.No. 70 (1993 Wageningen Pers, Wageningen, The Netherlands);

[0139] 2. Lallès, J. P., Tukur, H. M., Dréau, D. and Toullec, R.,Contribution of INRA to the Study of Antigenicity of Plant Protein Usedin Young Farm Animal Nutrition. In: Van Oort, M. G. and Tolman, G. H.:Antigenicity of Legume Proteins. TNO Communications. 25 pp (1992);

[0140] 3. Tukur, H. M., Lallès, J. P., Mathis, C., Caugant, I., andToullec, R., Digestion of Soybean Globulins, Glycinin, α-conglycinin andβ-conglycinin, in the Preruminant and the Ruminant Calf, Can. J. Anim.Sci., vol. 73, pp. 891-905 (December 1993);

[0141] 4. Lallès, J. P., Tukur, H. M., Toullec, R., and Miller, B. G.,Analytical Criteria for Predicting Apparent Digestibility of SoybeanProtein in Preruminant Calves, J. Dairy Sci., vol 79, pp 475-482 (1996);

[0142] 5. Tukur, H. M.; Lallès, J. P.; Plumb, G. W.; Mills, E. N. C.;

[0143] Morgan, M. R. A.; and Toullec, R., Investigation of theRelationship Between in Vitro Elisa Measures of Immunoreactive SoyGlobulins and in Vivo Effects of Soy Products, Journal of Agriculturaland Food Chemistry, 44 (8) pp. 2155-2161 (1996);

[0144] 6. Lallès, J. P., Tukur, H. M., Salgado, P., Mills, E. N. C.,Morgan, M. R. A., Quillien, L., Levieux, D., and Toullec, R.,Immunochemical Studies on Gastric and Intestinal Digestion of SoybeanGlycinin and Beta-conglycinin in Vivo, Journal of Agricultural and FoodChemistry, 47 (7) pp. 2797-2806 (July, 1999);

[0145] 7. Lallès, J. P.; Tukur, H. M.; and Toullec, R., ImmunochemicalTests for Measuring Glycinin and Beta-conglycinin Concentrations inSoyabean Products. Predictive Value for Nitrogen Digestibility andSoyabean Immunogenicity in the Calf, Annales de Zootechnie (Paris), 46(3), pp 193-205 (1997), CAB Accession Number: 981400459, BIOSIS NO.:199799684390;

[0146] 8. Lallès J. P.; Tukur H. M.; and Toullec, R., ImmunochemicalTests for the Determination of Glycinin and Beta-conglycinin Levels inSoya Products for Calf Milk Replacers, EAAP Publication, vol. 81, pp.243-244 (1996), BIOSIS NO.: 199699169689;

[0147] 9. Tukur, H. M., Pardal, P. B., Formal, M., Toullec, R., Lallès,J. P., and Guilloteau, P. Digestibility, Blood Levels of Nutrients andSkin Responses of Calves Fed Soyabean and Lupin Proteins, ReproductionNutrition Development vol. 35 (1) pp. 27-44 (1995);

[0148] 10. Toullec, R.; Lallès, J. P.; and Tukur, H. M., BiochemicalCharacteristics and Apparent Digestibility of Nitrogen in Soyabeans inPre-ruminant Calves (Original Title: Caracteristiques Biochimiques etDigestibilite Apparente Des Matieres Azotees De Soja Chez Le VeauPreruminant), ISBN: 2-84148-004-6, pp.229-232, (1994 Institut del'Elevage, Paris, France);

[0149] 11. Lallès, J. P., Tukur, H. M., and Toullec, R., Assessment ofthe Antigenicity of Soya Products for Calf Milk Replacers: WhichImmunochemical Tests to Use? (Evaluation De L'antigenicite Des Produitsdu Soja Destines Aux Aliments D'allaitement Pour Veaux: Quels TestsImmunochimiques Utiliser?), p. 135 in Proceedings of the 2nd meeting“Rencontres Autour Des Recherches Sur Les Ruminants” of the InstitutNational de la Recherche Agronomique, held in Paris (France), on Dec. 13and 14, 1995, (December, 1995, Institut de L'Elevage, Paris, France),ISBN: 2-84148-016-X;

[0150] 12. Toullec, R., Lallès, J. P., and Tukur, H. M., RelationshipsBetween Some Characteristics of Soybean Products and Nitrogen ApparentDigestibility in Preruminant Calves (Caracteristiques Biochimiques etDigestibilite Apparente Des Matieres Azotees De Soja Chez Le VeauPreruminant), pp. 229-232 of the Proceedings of the first meeting“Rencontres autour des recherches sur les ruminants”. of the InstitutNational de la Recherche Agronomique, held in Paris (France), on Dec. 1and 2, 1994, (December, 1994, Institut de l'Elevage, Paris, France),ISBN: 2-84148-004-6;

[0151] 13. Lallès, J. P. and Toullec, R., Soybean Products in MilkReplacers for Farm Animals: Processing, Digestion and Adverse Reactions,CAB Accession Number: 991411987;

[0152] 14. Lallès, J. P., Heppell, L. M. J., Sissons, J. W., andToullec, R., Antigenicity of Dietary Protein from Soyabean Meal and Peasin the Dairy Calf Throughout Weaning, CAB Accession Number: 920451145;

[0153] 15. Dreau, D., Larre, C., and Lalles, J. P. Semi-quantitativePurification and Assessment of Purity of Three SoybeanProteins—Glycinin, Beta-conglycinin and Alpha-conglycinin—by Sds-pageElectrophoresis, Densitometry and Immunoblotting, Journal of FoodScience and Technology, India, vol. 31 (6), pp. 489-493 (1994), ISSN:0022-1155;

[0154] 16. Heppell, L. M. J., Sissons, J. W., and Pedersen, H. E., AComparison of the Antigenicity of Soybean-based Infant Formulas, BritishJournal of Nutrition, vol. 58 (3), pp.393-404 (1987);

[0155] 17. Sissons, J. W. and Thurston, S. M., Survival of DietaryAntigens in the Digestive Tract of Calves Intolerant to SoyabeanProducts, Research in Veterinary Science vol. 37 (2): pp. 242-246(1984);

[0156] 18. Sissons, J. W., Nyrup, A., Kilshaw, P. J.; and Smith, R. H.,Ethanol Denaturation of Soybean Protein Antigens, Journal of the Scienceof Food and Agriculture, vol. 33 (8): pp. 706-710 (1982);

[0157] 19. Kilshaw, P. J., and Sissons, J. W., Gastrointestinal Allergyto Soyabean Protein in Preruminant Calves. Allergenic Constituents ofSoyabean Products, Research in Veterinary Science, vol. 27 (3): pp.366-371 (1979);

[0158] 20. Heppel, L. M. J., Determination of milk protein Denaturationby an Enzyme-Linked Immunosorbent Assay, Pages 115-123 in Morris, B. A.and Clifford, M. N., eds., Immunoassays in Food Analysis (1985 ElsevierApplied Science publishers, London, England); and

[0159] 21. Bush, R. S., Toellec, R., Caugant, I., and Guilloteau, P.,Effects of Raw Pea Flour on Nutrient Digestibility and Immune Responsesin the Preruminant Calf, J. Dairy Sci., vol. 75, pp. 3539-3552 (1992).

[0160] 22. Perez, M. D., Mills, E N Clare, Lambert, N., Johnson, I. T.,and Morgan, M. R. A., The Use of Anti-Soya Globulin Antsera inInvestigating Soya Digestion In Vivo, J. of the Science of Food andAgriculture, vol. 80, pp. 513-521 (2000).

EXAMPLES

[0161] The present invention is more particularly described in thefollowing examples which are intended as illustrations only sincenumerous modifications and variations within the scope of the presentinvention will be apparent to those skilled in the art.

Example 1

[0162] This example demonstrates the effectiveness of the process of thepresent invention for substantially enhancing the Protein DispersabilityIndex (PDI) of soy flakes and for substantially decreasing the contentof antigenic proteins, such as glycinin and β-conglycinin, in soy flakesthat are treated in accordance with the process of the presentinvention. In Example 1, 110 gallons (416.4 liters) {968 pounds (439.1kilograms)} of warm water were added to a 240 gallon (908 liter) tank(subsequently referred to as a “batch reactor”). The batch reactor wasequipped with an agitator. The batch reactor was also equipped with ajacket for accommodating steam or hot water circulation to maintain orchange the temperature of the contents of the batch reactor. With thewater at a temperature of about 50° C., 300 pounds (136.1 kilograms) ofsoy flakes were added to the warm water in the batch reactor under slowagitation to form a homogenous slurry of the soy flakes and water. Thesoy flakes were obtained from Harvest States Oilseed Processing &Refining of Mankato, Minn. After addition of the soy flakes wascompleted, hot water was circulated through the jacket of the batchreactor to raise the temperature of the slurry to about 53° C.

[0163] After formation of the slurry of soy flakes and water, theinitial pH of the slurry was about 6.35 standard pH units. About 12liters of a solution of 10 weight percent NaOH, based upon the totalweight of the sodium hydroxide solution, was added to the slurry toadjust the pH of the slurry to about 9.00 standard pH units. The amountof sodium hydroxide solution that was added boosted the pH of the slurryhigher than desired. Therefore, with the slurry still under agitation,about four liters of an aqueous acid solution containing about 10 weightpercent hydrochloric acid, based upon the total weight of the aqueousacid solution, was gradually added to the agitated slurry until the pHof the slurry was reduced to about 8.48 standard pH units.

[0164] About 3 pounds (1360 grams) {about 1500 milliliters} ofMULTIFECT® P-3000 enzyme composition were then added to the slurry inthe batch reactor while agitating the slurry. Thus, the MULTIFECT®P-3000 enzyme composition was added to the slurry at a ratio of aboutone pound (454 grams) of MULTIFECT® P-3000 enzyme composition per onehundred pounds (45.35 kilograms) of soy flakes. The MULTIFECT® P-3000enzyme composition, which is a dark amber colored liquid, was obtainedfrom Genencor International, Inc. of Santa Clara, Calif. The addition ofthe MULTIFECT® P-3000 enzyme composition initiated an enzymatichydrolysis reaction that was allowed to continue in the batch reactorfor a period of about two hours while maintaining the slurry at atemperature ranging from about 53° C. to about 55° C. and whilemaintaining mild agitation of the slurry.

[0165] No caustic or acid was added to the slurry during the enzymatichydrolysis, and the pH of the slurry was observed to drop to about 7.07standard pH units after the two-hour period of enzymatic hydrolysis. Atthe end of the two hour enzymatic hydrolysis period, steam was passedthrough the jacket of the batch reactor and the slurry was heated toabout 85° C. to inactivate the alkaline proteolytic enzyme. Temperatureand pH details during the two hour period of enzymatic hydrolysis andtemperature details during the heating to inactivate the enzymes areprovided below in Table 1: TABLE 1 Time Temp Description (minutes) pH (°C.) Start of Enzymatic Hydrolysis 0 8.48 53.0 20 55.3 60 7.13 54.6 857.08 54.0 Start of Heating to Inactivate Enzymes 117 7.07 53.2 123 57.6131 64.4 Target Enzyme Inactivation Temp. Achieved 150 85.0 EnzymeInactivation Completed 155 85.0

[0166] Heating of the slurry to inactivate the alkaline proteolyticenzyme was begun at time 117 (minutes). The slurry was held at about 85°C. for about 5 minutes.

[0167] After enzyme inactivation was completed, the slurry was thenpumped from the batch reactor to a pair of 120 gallon (454 liter)storage tanks equipped with agitators. Cold water was circulated throughthe jacket of the batch reactor during the transfer of the slurry to thestorage tanks. Also, en route to the storage tanks, the slurry waspassed through a COMITROL® Model No. 1700 processor to ensure that anyfibrous material in the slurry was broken apart prior to drying. Aftertransfer of the slurry through the COMITROL® processor and to thestorage tanks was completed, 10 gallons (37.8 liters) of hot tap waterwas added to the slurry in one of the storage tanks and 15 gallons (56.8liters) of hot tap water was added to the slurry in the other of thestorage tank to facilitate subsequent spray drying. After hot wateraddition was completed, the diluted slurry in each of the storage tankswas introduced into a vertical spray dryer, supplied by C. E. Rogers Co.of Northville, Mich., to produce spray dried soy powder. The recoveryrate for the processing described above in this example was about 90.7%,since 300 pounds (136.1 kilograms) of soy flakes were introduced intothe batch reactor, and 272 pounds (123.4 kilograms) of spray dried soypowder were recovered from the spray dryer.

[0168] Samples of the soy flakes that were added as feed to the batchreactor and samples of the spray dried soy powder were analyzed forvarious properties. The result of these properties for the soy flakesand for the spray dried soy powder are provided in Table 2 below: TABLE2 PROPERTY SOY FLAKES SPRAY DRIED Dry matter (weight %) 96.25 94.77Organic matter (weight %, based on dry matter weight) 92.41 89.53 Ash(weight %, based on dry matter weight) 7.59 10.47 Crude protein (weight%, based on dry matter weight) 49.41 45.96 CP (weight %, based onorganic matter weight) 53.46 51.33 Soluble protein (weight %, based oncrude protein weight) 66.50 85.10 Immunoreactive glycinin (mg/g CrudeProtein) 469 227.3 Immunoreactive β-conglycinin (mg/g Crude Protein) 3020.046 Glycinin + β-conglycinin (mg/g Crude Protein) 771 227

[0169] The weight percent of dry matter, organic matter, ash, crudeprotein, and crude protein in the soy flakes and in the spray dried soypowder were determined in accordance with the procedures for thesevariables set forth above in the Property Determination &Characterization Technique section. The glycinin and β-conglycininconcentrations in the soy flakes and in the spray dried soy powder weredetermined in accordance with the Glycinin and β-conglycininDeterminations technique that is described above in the PropertyDetermination & Characterization Techniques section.

[0170] The results shown in Table 2 demonstrate that, even though thesoy flakes used as feed in this example contained little, if any,denatured protein, enzymatic hydrolysis in accordance with the presentinvention was nonetheless effective to increase the concentration ofsoluble protein by about 28 percent in the spray dried soy powder, ascompared to the concentration of soluble protein in the soybean flakesused as feed. Also, the enzymatic hydrolysis procedure decreased theglycinin concentration by about 51.5 percent in the spray dried soypowder, as compared to the glycinin concentration in the soy flakes usedas feed. Additionally, the enzymatic hydrolysis reduced theconcentration of β-conglycinin by about 99.9 percent in the spray driedsoy powder, as compared to the concentration of β-conglycinin in the soyflakes used as feed. Consequently, the enzymatic hydrolysis waseffective to reduce the concentrations of the principal antigenicproteins (glycinin plus β-conglycinin) by about 70.5 percent in thespray dried soy powder, as compared to the concentrations of theprincipal antigenic proteins (glycinin plus β-conglycinin) in the soyflakes used as feed.

[0171] Additionally, the soy flakes and the spray dried soy powder wereanalyzed by high pressure liquid chromatography (HPLC) to detect anyshift in molecular weight distribution of protein fragments in the spraydried soy powder versus the soy flakes that were used as feed. The highpressure liquid chromatography analysis was conducted in accordance withthe procedure set forth above in the Property Determination &Characterization Techniques section. The HPLC results for the soy flakesare provided in the graph of FIG. 1, and the HPLC results for the spraydried soy powder are provided in the graph of FIG. 2. Details showingthe variables used in determining the peak areas and the peak areavalues of each of the nine peaks shown in the graph of FIG. 1 and forthe eight peaks shown in the graph of FIG. 2 are provided below inTables 3 and 4, respectively. TABLE 3 Peak Retention Time Area Height %% No. (min) (uV × sec) (uV) Area Height 1 6.893 1998958 54950 9.47 10.002 7.577 747017 34379 3.54 6.25 3 8.493 3503407 80267 16.60 14.60 4 9.3931717441 43824 8.14 7.97 5 10.327 710860 14281 3.37 2.60 6 11.727 1773665774 0.84 1.05 7 12.377 514409 14148 2.44 2.57 8 13.293 538726 235032.55 4.28 9 14.243 11193027 278605 53.04 50.68

[0172] TABLE 4 Peak Retention Time Area Height % % No. (min) (uV × sec)(uV) Area Height 1 6.467 304828 12772 1.06 2.62 2 6.883 695246 172432.41 3.53 3 8.967 2113479 37925 7.33 7.77 4 10.367 558318 9941 1.94 2.045 11.483 778394 17172 2.70 3.52 6 14.400 23445185 365929 81.29 74.97 715.600 897585 25413 3.11 5.21 8 17.450 49551 1728 0.17 0.35

[0173] The graphs of FIGS. 1 and 2 maybe readily interpreted when it isrecognized that protein fragments with larger molecular weights show upearlier during the HPLC scan in peaks with shorter retention times andprotein fragments with smaller molecular weights show up later duringthe HPLC scan in peaks with longer retention times. Thus, in the graphof FIG. 2, as compared to the graph of FIG. 1, there was a shift tolarger peak areas at higher retention time as compared to peak areas atsimilar retention times in the graph of FIG. 1. This demonstrates thatthe spray dried soy powder, as represented in the graph of FIG. 2,contained protein fragments with a smaller molecular weight average andprofile as compared to the soy flakes depicted in the graph of FIG. 1.This correlates well with the substantially enhanced soluble proteinconcentration in the spray dried soy powder, as compared to the solubleprotein concentration in the soy flakes.

Example 2

[0174] This example demonstrates the effectiveness of the process of thepresent invention for substantially enhancing the protein dispersabilityindex (PDI) of defatted soy flour with a PDI of about 20 that containeda substantial amount of denatured protein. This example demonstrates theeffectiveness of the process of the present invention for substantiallydecreasing the concentration of antigenic proteins, such as glycinin andP-conglycinin, in the 20 PDI defatted soy flour.

[0175] In this example, the 20 PDI soy flour was HONEYSOY® 20 PDI soyflour that was obtained from Harvest States Oilseed Processing &Refining of Mankato, Minn. The 20 PDI soy flour was combined with warmtap water (50° C.) in several batches at the rate of about 2 pounds (907grams) of 20 PDI flour per gallon (3.78 liters) of warm tap water. Eachbatch of 20 PDI soy flour was processed in a Model No. LTDW liquefierobtained from Breddo Likwifier of Kansas City, Kans. to liquity andslurry the mixture of 20 PDI soy flour and water.

[0176] Each batch of liquified soy flour/water slurry was transferredfrom the liquefier into a 250 gallon (946 liter) batch reactor that wasidentical to the 250 gallon (946 liter) batch reactor described inExample 1 above. A total of 325 pounds (147.4 kilograms) of the 20 PDIsoy flour was combined with a total of 162.5 gallons (615.1 liters) ofwater in the soy flour/water slurry that was placed in the batchreactor. After addition of the 20 PDI soy flour and water to the batchreactor was completed, hot water was circulated through the jacket ofthe batch reactor to raise the temperature of the soy flour/water slurryto about 56.7° C. The initial pH of the soy flour/water slurry was about6.67 standard pH units, and the initial Brookfield viscosity of the soyflour/water slurry was about 1240 centipoise at 56.7° C.

[0177] About 16 liters of a aqueous solution of 10 weight percent NaOHin water, based on the total weight of the sodium hydroxide solution,was added to the soy flour/water slurry to adjust the pH of the soyflour/water slurry to about 9.05 standard pH units. Then, about 3.3pounds (about 1.5 kilograms) {about 1.65 liters} of the MULTIFECT®P-3000 enzyme composition was added to the soy flour/water slurry in thebatch reactor. Thus, the MULTIFECT® P-3000 enzyme composition was addedat a ratio of about one pound (about 454 grams) of the MULTIFECT® P-3000enzyme composition per one hundred pounds (45.35 kilograms) of 20 PDIsoy flour. After addition of the enzyme solution, the temperature of thesoy flour/water slurry was determined to be about 56.7° C. and theBrookfield viscosity of the soy flour/water slurry was determined to beabout 1900 centipoise at the 56.7° C. slurry temperature.

[0178] The enzymatic hydrolysis reaction triggered by addition of theMULTIFECT® P-3000 enzyme composition was allowed to continue in thebatch reactor for a period of about 2 hours while maintaining the soyflour/water slurry at a temperature ranging from about 56.7° C. to about60° C. No caustic or acid was added to the slurry during the enzymatichydrolysis, and the pH of the slurry was observed to drop to about 7.58standard pH units after the two-hour period of enzymatic hydrolysis. ThepH, viscosity, and temperature of the soy flour/water slurry at varioustimes during the two-hour enzymatic hydrolysis reaction are shown inTable 5 below: TABLE 5 Time Viscosity Temp Description (minutes) pH (cp)(° C.) Start of Enzymatic Hydrolysis 0 9.05 1900 56.7 5 8 200 57.3 157.9 150 58.5 30 7.77 110 58.6 60 7.47 110 59 90 7.64 120 59.3 End ofEnzymatic Hydrolysis 120 7.58 80 60

[0179] Thus, the enzymatic hydrolysis reaction caused the Brookfieldviscosity of the slurry to fall from about 1900 centipoise, measured at56.7° C., to about 80 centipoise, measured at about 60° C.

[0180] After the two hour enzymatic hydrolysis period, steam was sentthrough the jacket of the batch reactor to inactivate the alkalineproteolytic enzymes. As the slurry was being heated, several Brookfieldviscosity determinations were made. At 70° C., the Brookfield viscosityof the viscosity was found to be about 110 centipoise, at 80° C. theBrookfield viscosity of the slurry was found to be about 280 centipoise,and at 90° C. the Brookfield viscosity of the slurry was found to beabout 440 centipoise. After reaching 90° C., the slurry was held at thetemperature of about 90° C. to about 95° C. for a period of about 10minutes to complete inactivation of the alkaline proteolytic enzyme.After the 10 minute enzyme inactivation period, the Brookfield viscosityof the slurry was determined to be about 420 centipoise at 90° C. andabout 850 centipoise at room temperature (about 70° F.).

[0181] After enzyme inactivation was completed, the slurry was cooledand comminution in similar fashion to the cooling and comminutiondescribed in Example 1 and was thereafter spray dried using a verticalspray dryer obtained from C. E. Rogers Co. to produce spray dried soyflour. The recovery rate for the processing described above in thisexample was about 93.5%, since 325 pounds (147.42 kilograms) of 20 PDIsoy flour were introduced into the batch reactor, and 304 pounds (137.9kilograms) of spray dried soy flour were recovered from the spray dryer.

Example 3

[0182] This example is similar to Example 2 and consequentlydemonstrates the capabilities of the process of the present inventionfor substantially enhancing the Protein Dispersability Index (PDI) ofdefatted soy flour with a PDI of about 20 that contains a substantialamount of denatured protein and for substantially decreasing the contentof antigenic proteins, such as glycinin and β-conglycinin in the 20 PDIdefatted soy flour.

[0183] HONEYSOY® 20 PDI soy flour was used as the feed material in thisexample as in Example 2. Slurry containing the same ratio of 20 PDI soyflour to water was prepared and liquified as described in Example 2 andplaced in a batch reactor that was identical to the batch reactor usedin Example 2. A total of 175 pounds (79.4 kilograms) of 20 PDI soy flourwas combined with a total of 87.5 gallons (331.3 liters) of water in thesoy flour/water slurry that was placed in the batch reactor. Afteraddition of the 20 PDI soy flour and water to the batch reactor wascompleted, hot water was circulated through the jacket of the batchreactor to raise the temperature of the soy flour/water slurry to about54° C. The initial pH of the slurry in the batch reactor was about 6.56standard pH units, and the initial Brookfield viscosity of the slurrywas about 2800 centipoise at a slurry temperature of about 54° C. Aboutone hour after preparation, while still being agitated, the pH of theslurry was observed to have dropped to about 6.2 standard pH units.

[0184] About 8.5 liters of the 10 weight percent NaOH aqueous solutionwas added to the slurry to adjust the pH of the slurry to about 9.06standard pH units. Then, about 1.6 pounds (725.7 grams) {about 0.8liters} of the MULTIFECT® P-3000 enzyme composition was added to the soyflour/water slurry in the batch reactor. Thus, the MULTIFECT® P-3000enzyme composition was added at a ratio of about one pound (454 grams)of the MULTIFECT® P-3000 enzyme composition per one hundred pounds(45.35 kilograms) of 20 PDI soy flour. After addition of the enzymesolution, the temperature of the soy flour/water slurry was determinedto be about 53.9° C. and the Brookfield viscosity of the soy flour/waterslurry was determined to be about 1730 centipoise at the 53.9° C. slurrytemperature.

[0185] The enzymatic hydrolysis reaction triggered by an addition of theMULTIFECT® P-3000 enzyme composition was allowed to continue in thebatch reactor for a period of about 2 hours while maintaining the soyflour/water slurry at a temperature ranging from about 54° C. to about60° C. No caustic or acid was added to the slurry during the enzymatichydrolysis, and the pH of the slurry was observed to drop to about 7.21standard pH units after the two-hour period of enzymatic hydrolysis. ThepH, viscosity, and temperature of the soy flour/water slurry at varioustimes during the two-hour enzymatic hydrolysis reaction are shown inTable 6 below: TABLE 6 Time Viscosity Temp Description (minutes) pH(centipoise) (° C.) Start of Enzymatic Hydrolysis 0 9.06 1730 53.9 57.58 220 54.8 60 7.31 80 58.8 90 7.26 60 59.2 End of EnzymaticHydrolysis 120 7.21 60 59.2

[0186] Thus, the enzymatic hydrolysis reaction caused the Brookfieldviscosity of the slurry to fall from about 1730 centipoise, measured at53.9° C., to about 60 centipoise, measured at about 59.2° C.

[0187] After the two-hour enzymatic hydrolysis period, steam was enteredinto the jacket of the batch reactor to inactivate the alkalineproteolytic enzymes. After reaching 90° C. the soy flour/water slurrywas held at the temperature of about 90° C. to about 95° C. for a periodof about 10 minutes to complete inactivation of the alkaline proteolyticenzyme.

Discussion of Results for Examples 2 and 3

[0188] Examples 2 and 3 each used the same 20 PDI defatted soy flour asthe feed material upon which enzymatic hydrolysis was conducted.Examples 2 and 3 each used the same ratio of 20 PDI defatted soy flourto water in the slurry that was enzymatically hydrolyzed. Examples 2 and3 each used the same alkaline agent and arrived at approximately thesame pH both before and after addition of the alkaline agent and theenzyme solution. Also, Examples 2 and 3 each used the same MULTIFECT®P-3000 enzyme composition, and the same weight ratio of the MULTIFECT®P-3000 enzyme composition to 20 PDI soy flour ratio was used in bothExamples 2 and 3. A graph that is included as FIG. 3 illustrates how thepH profiles of the slurry during the enzymatic hydrolysis reactionstrack in very similar fashion for both Examples 2 and 3. This graph ofFIG. 3 also illustrates how the viscosity profiles of the slurry duringthe enzymatic hydrolysis reactions track in very similar fashion forboth Examples 2 and 3. It is noted that the viscosities plotted in FIG.3 were not corrected to a standard temperature, but were insteaddetermined at the temperature of the slurry at the time of sampling.

[0189] Samples of the 20 PDI soy flour used as the feed material to behydrolyzed in Examples 2 and 3 above were collected and blended inpreparation for analysis. The blended 20 PDI soy flour sample andsamples of the spray dried soy flour obtained in Examples 2 and 3 wereanalyzed for various properties. The results of these analyses areprovided in Table 7 below: TABLE 7 EXAMPLE 2 EXAMPLE 3 SPRAY SPRAY SOYDRIED SOY DRIED SOY PROPERTY FLOUR FLOUR FLOUR Dry matter (weight %)94.0 94.5 93.9 Organic matter (weight %, based on dry matter weight)93.3 92.1 92.3 Ash (weight %, based on dry matter weight) 6.7 7.9 7.7Crude protein (weight %, based on dry matter weight) 48.6 48.0 47.3 CP(weight %, based on organic matter weight) 52.1 52.1 51.2 Solubleprotein (weight %, based on crude protein weight) 20.8 78.4 81.5Immunoreactive glycinin (mg/g Crude Protein) 71 7.5 21 Immunoreactiveβ-conglycinin (mg/g Crude Protein) 32 0 0 Glycinin + β-conglycinin (mg/gCrude Protein) 103 7.5 21

[0190] The weight percent of dry matter, organic matter, ash, crudeprotein, and crude protein in the 20 PDI soy flour and in the spraydried soy flour were determined in accordance with the procedures forthese variables set forth above in the Property Determination &Characterization Technique section. The glycinin and β-conglycininconcentrations in the 20 PDI soy flour and in the spray dried soy flourwere determined in accordance with the Glycinin and β-conglycininDeterminations technique that is described above in the PropertyDetermination & Characterization Techniques section.

[0191] The results shown in Table 7 demonstrate that the enzymatichydrolysis procedures that were carried out in Examples 2 and 3 wereeach effective to dramatically improve the PDI of the blended 20 PDI soyflour from a PDI of about 20 all the way up to a PDI on the order ofabout 80 for the spray dried soy flour, specifically, a PDI of 78.4 forExample 2 and a PDI of 81.5 for Example 3. Thus, the enzymatichydrolysis of Example 2 improved the PDI in the spray dried soy flour byabout 277 percent, as compared to the 20 PDI soy flour, whereas theenzymatic hydrolysis of Example 3 improved the PDI in the spray driedsoy flour by about 292 percent, as compared to the 20 PDI soy flour.These dramatic increases in the PDI values for soy flours treated inaccordance with the present invention graphically illustrate the abilityof the present invention to improve solubilities of vegetable proteinmatter, such as those containing denatured soy proteins.

[0192] Also, the enzymatic hydrolysis procedures of Examples 2 and 3effected dramatic decreases in the concentration of one antigenicprotein, glycinin. In Example 2, the glycinin concentration in the spraydried soy flour was about 89 percent less than the glycinin level in the20 PDI soy flour, whereas in Example 3 the decrease in glycininconcentration for the spray dried soy flour was a more modest 70percent, as compared to the glycinin concentration in the 20 PDI soyflour. The inventive enzymatic hydrolysis process was even more dramaticin its effectiveness at treating another antigenic protein, namelyβ-conglycinin. More specifically, in Examples 2 and 3, the enzymatichydrolysis procedure was able to completely eliminate any β-conglycinincontent in the spray dried soy flour produced in Examples 2 and 3, eventhough the 20 PDI soy flour used as feed in these examples had aβ-conglycinin concentration of 32 milligrams per gram of crude protein.Consequently, in Example 2, the enzymatic hydrolysis was effective toreduce the overall concentration of the principal antigenic proteins(glycinin plus β-conglycinin) by nearly 90 percent in the spray driedsoy powder, as compared to the concentration of the principal antigenicproteins in the 20 PDI soy flour. Likewise, the enzymatic hydrolysis waseffective to reduce the concentration of the principal antigenicproteins (glycinin plus β-conglycinin) by about 80 percent in the spraydried soy flour of Example 3, as compared to the concentration of theprincipal antigenic proteins in the 20 PDI soy flour.

[0193] Samples of a blend of the 20 PDI soy flour used in Examples 2 and3 as the feed material and samples of a blend of the spray dried soyflour produced in Examples 2 and 3 were analyzed by high pressure liquidchromatography (HPLC) to detect any shift in the spray dried soy flourtoward protein fragments with smaller molecular weights versus the 20PDI soy flour. The high pressure liquid chromatography analysis wasconducted in accordance with the procedure set forth above in theProperty Determination & Characterization Techniques section. The HPLCresults for the blend of 20 PDI soy flour used as feed in Examples 2 and3 are provided in the graph of FIG. 4, and the HPLC results for blend ofspray dried soy flour from Examples 2 and 3 are provided in the graph ofFIG. 5.

[0194] The graphs of FIGS. 4 and 5 may be readily interpreted when it isrecognized that protein fragments with larger molecular weights show upearlier during the HPLC scan in peaks with shorter retention times andprotein fragments with smaller molecular weights show up later duringthe HPLC scan in peaks with longer retention times. Thus, in the graphof FIG. 5, as compared to the graph of FIG. 4, there was a shift tolarger peak areas at higher retention times as compared to peak areas atsimilar retention times in the graph of FIG. 4. This demonstrates thatthe blend of spray dried soy flours produced in Examples 2 and 3, asrepresented in the graph of FIG. 5, contained protein fragments with asmaller molecular weight average and profile, as compared to the sampleof blended 20 PDI soy flour feed material from Examples 2 and 3, asdepicted in the graph of FIG. 4. This correlates well with thesubstantially enhanced soluble protein concentration (increased PDI) inthe spray dried soy flour samples of Examples 2 and 3, as compared tothe soluble protein concentration in the 20 PDI soy flours used as feedin Examples 2 and 3.

[0195] The graphs of FIGS. 4 and 5, when subject to a regressionanalysis, further demonstrate the beneficial protein molecular weightreduction achieved by the process of the present invention.Specifically, this regression analysis revealed that the sample ofblended 20 PDI soy flour feed material from Examples 2 and 3, asdepicted in the graph of FIG. 4, includes mostly protein fragments witha molecular weight size ranging from about 123 kilodaltons to about 394kilodaltons. On the other hand, regression analysis revealed that theblend of spray dried soy flours produced in Examples 2 and 3, asrepresented in the graph of FIG. 5, includes mostly protein fragmentswith a molecular weight size below about 2400 Daltons, with the actualrange extending from about 200 Daltons to about 2400 Daltons for thevast majority of the protein fragments.

[0196] Although the present invention has been described with referenceto preferred embodiments, workers skilled in the art will recognize thatchanges may be made in form and detail without departing from the spiritand scope of the invention.

1. A method of treating a proteinaceous material having a firstconcentration of β-conglycinin, the method comprising: combining theproteinaceous material with an enzyme to form a reaction mixture, thereaction mixture initially having a pH of at least about 7.0 standard pHunits; allowing the enzyme to hydrolyze β-conglycinin present in thereaction mixture to form a proteinaceous intermediate; and inactivatingthe enzyme present in the reaction mixture after a reaction period toform a proteinaceous product, the proteinaceous product having a secondconcentration of β-conglycinin, the second concentration ofβ-conglycinin being at least 99 percent less than the firstconcentration of β-conglycinin.
 2. The method of claim 1 wherein thereaction period begins when the reaction mixture is formed and ends whenenzyme inactivation is initiated, the reaction period being about fiveminutes to about two hours.
 3. The method of claim 2 wherein thereaction mixture initially has a pH of at least about 8.5 standard pHunits.
 4. The method of claim 3 wherein the method is effective toprovide the second concentration of β-conglycinin that is at least 99percent less than the first concentration of β-conglycinin when no pHadjustment is made during the reaction period after initiation of theenzymatic hydrolysis.
 5. The method of claim 1 wherein the reactionmixture initially has a pH greater than about 8.5 standard pH units. 6.The method of claim 1 wherein the second concentration of β-conglycininis about 100 percent less than the first concentration of β-conglycinin.7. The method of claim 1 wherein only a single stage hydrolysis reactionoccurs during the reaction period.
 8. A method of treating aproteinaceous material having a first concentration of glycinin, themethod comprising: combining the proteinaceous material with an enzymeto form a reaction mixture, the reaction mixture initially having a pHof at least about 7.0 standard pH units; allowing the enzyme tohydrolyze glycinin present in the reaction mixture to form aproteinaceous intermediate; and inactivating the enzyme present in thereaction mixture after a reaction period to form a proteinaceousproduct, the proteinaceous product having a second concentration ofglycinin, the second concentration of glycinin being at least about 50percent less than the first concentration of glycinin.
 9. The method ofclaim 8 wherein the reaction period begins when the reaction mixture isformed and ends when enzyme inactivation is initiated, the reactionperiod being about five minutes to about two hours.
 10. The method ofclaim 9 wherein the reaction mixture initially has a pH of at leastabout 8.5 standard pH units.
 11. The method of claim 10 wherein themethod is effective to provide the second concentration of glycinin thatis at least about 50 percent less than the first concentration ofglycinin when no pH adjustment is made during the reaction period afterinitiation of the enzymatic hydrolysis.
 12. The method of claim 8wherein the reaction mixture initially has a pH greater than about 8.5standard pH units.
 13. The method of claim 8 wherein the secondconcentration of glycinin is at least about 70 percent less than thefirst concentration of glycinin.
 14. The method of claim 8 wherein onlya single stage hydrolysis reaction occurs during the reaction period.15. A method of treating a proteinaceous material having a first ProteinDispersability Index, the method comprising: combining the proteinaceousmaterial with an enzyme to form a reaction mixture, the reaction mixtureinitially having a pH of at least about 7.0 standard pH units; allowingthe enzyme to hydrolyze protein present in the reaction mixture to forma proteinaceous intermediate; and inactivating the enzyme present in thereaction mixture after a reaction period to form a proteinaceousproduct, the proteinaceous product having a second ProteinDispersability Index, the second Protein Dispersability Index being atleast about 20 percent greater than the first Protein DispersabilityIndex.
 16. The method of claim 15 wherein the first ProteinDispersability Index is at least about 60 percent.
 17. The method ofclaim 15 wherein the first Protein Dispersability Index is about 20percent, or less, and the second Protein Dispersability Index is atleast about 70 percent.
 18. The method of claim 15 wherein theproteinaceous material has an average protein molecular weight in therange of about 125 kilodaltons to about 440 kilodaltons and theproteinaceous product has an average protein molecular weight of about2500 Daltons or less.
 19. The method of claim 15 wherein the reactionperiod begins when the reaction mixture is formed and ends when enzymeinactivation is initiated, the reaction period being about five minutesto about two hours.
 20. The method of claim 19 wherein the reactionmixture initially has a pH of at least about 8.5 standard pH units. 21.The method of claim 20 wherein the proteinaceous material has an averageprotein molecular weight in the range of about 125 kilodaltons to about440 kilodaltons and the proteinaceous product has an average proteinmolecular weight of about 5000 Daltons or less.
 22. The method of claim20 wherein the first Protein Dispersability Index is about 20 percent,or less, the method effective to make the second Protein DispersabilityIndex at least about 70 percent when no pH adjustment is made during thereaction period after initiation of the enzymatic hydrolysis.
 23. Themethod of claim 15 wherein the reaction mixture initially has a pHgreater than about 8.5 standard pH units.
 24. The method of claim 23wherein the first Protein Dispersability Index is about 20 percent, orless, and the second Protein Dispersability Index is at least about 70percent.
 25. The method of claim 15 wherein only a single stagehydrolysis reaction occurs during the reaction period.
 26. A method oftreating a proteinaceous material, the method comprising: combining theproteinaceous material with water to form a slurry; adjusting the pH ofthe slurry to greater than about 8.5 standard pH units; combining analkaline proteolytic enzyme with the slurry; and permitting the alkalineproteolytic enzyme to hydrolyze protein contained in the slurry to forma proteinaceous product.
 27. The method of claim 26 wherein the pH ofthe slurry is adjusted to a pH within the range of about 9.0 standard pHunits to about 9.5 standard pH units.
 28. The method of claim 26 whereinonly a single stage hydrolysis reaction occurs in the slurry and no pHadjustment is made to the slurry after the enzyme is combined with theslurry.
 29. A method of treating a proteinaceous material, the methodcomprising: combining the proteinaceous material with an enzyme to forma reaction mixture, the reaction mixture initially having a pH of atleast about 7.0 standard pH units; allowing the enzyme to hydrolyzeprotein present in the reaction mixture to form a proteinaceousintermediate; and inactivating the enzyme present in the reactionmixture after a reaction period to form a proteinaceous product, theenzyme derived from a genetically modified strain of bacteria belongingto the species subtilis of the genus Bacillus.
 30. The method of claim29 wherein the proteinaceous material has a first concentration ofβ-conglycinin and the proteinaceous product has a second concentrationof β-conglycinin, the second concentration of β-conglycinin being atleast 99 percent less than the first concentration of β-conglycinin. 31.A method of treating a proteinaceous material, the method comprising:combining the proteinaceous material with an enzyme to form a reactionmixture, the reaction mixture initially having a pH of at least about7.0 standard pH units; allowing the enzyme to hydrolyze protein presentin the reaction mixture to form a proteinaceous intermediate; andinactivating the enzyme present in the reaction mixture after a reactionperiod to form a proteinaceous product, the enzyme belongs to thespecies subtilis of the genus Bacillus or belongs to the speciesamyloliquefaciens of the genus Bacillus.
 32. The method of claim 31wherein the proteinaceous material has a first concentration ofβ-conglycinin and the proteinaceous product has a second concentrationof β-conglycinin, the second concentration of β-conglycinin being atleast 99 percent less than the first concentration of β-conglycinin. 33.The method of claim 31 wherein the enzyme belongs to the speciesamyloliquefaciens of the genus Bacillus that is expressed by agenetically modified strain of bacteria belonging to the speciessubtilis of the genus Bacillus.
 34. A method of treating a proteinaceousmaterial, the method comprising: combining the proteinaceous materialwith an enzyme to form a reaction mixture, the reaction mixtureinitially having a pH of greater than about 8.5 standard pH units;allowing the enzyme to hydrolyze protein present in the reaction mixtureto form a proteinaceous intermediate; and inactivating the enzymepresent in the reaction mixture after a reaction period to form aproteinaceous product, the enzyme being either a (1) firstnaturally-occurring protease or (2) a recombinant protease, therecombinant protease directly or indirectly derived from a secondnaturally-occurring protease from any source or from anaturally-occurring peptide hydrolase from any source.
 35. The method ofclaim 34 wherein the proteinaceous material has a first concentration ofβ-conglycinin and the proteinaceous product has a second concentrationof β-conglycinin, the second concentration of β-conglycinin being atleast 99 percent less than the first concentration of β-conglycinin. 36.The method of claim 34 wherein the reaction mixture initially has a pHwithin the range of about 9.0 standard pH units to about 9.5 standard pHunits.
 37. The method of claim 34 wherein the enzyme is either anaturally-occurring serine protease or a recombinant serine protease.38. The method of claim 34 wherein the enzyme is either anaturally-occurring bacillus subtilisin or a recombinant bacillussubtilisin.
 39. The method of claim 34 wherein the enzyme is either (1)a naturally-occurring subtilisin secreted by B. licheniformis, B.amyloliquefaciens, or B. subtilis or (2) a recombinant subtilisin thatis directly or indirectly derived from a naturally-occurring subtilisinsecreted by B. licheniformis, B. amyloliquefaciens, or B. subtilis.